NICEATM Summary Review Document: Short Time Exposure Test … · 2020. 10. 19. · Short Time Exposure (STE) Test Method Summary Review Document Interagency Coordinating Committee

Short Time Exposure (STE) Test Method

Summary Review Document

Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)

National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)

National Institute of Environmental Health Sciences National Institutes of Health U.S. Public Health Service

Department of Health and Human Services

2013

National Toxicology Program P.O. Box 12233

Research Triangle Park, NC 27709

Short Time Exposure Summary Review Document

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Table of Contents

List of Tables ......................................................................................................................................... v List of Abbreviations and Acronyms ................................................................................................. vi Acknowledgements ............................................................................................................................. vii Executive Summary ............................................................................................................................. 1 1.0 Introduction and Background ................................................................................................... 5 2.0 STE Test Method Database ........................................................................................................ 7 3.0 STE Test Method Performance ............................................................................................... 30

3.1 GHS Classification System: STE Performance in a Top-Down Approach ................... 30 3.1.1 STE Discordant Results for GHS Classification in a Top-Down

Approach .......................................................................................................... 31 3.2 GHS Classification System: STE Performance in a Bottom-Up Approach ................... 33

3.2.1 STE Discordant Results for GHS Classification in a Bottom-Up Approach .......................................................................................................... 34

3.3 EPA Classification System: STE Performance in a Top-Down Approach .................... 36 3.3.1 STE Discordant Results for EPA Classification in a Top-Down

Approach .......................................................................................................... 37 3.4 EPA Classification System: STE Performance in a Bottom-Up Approach ................... 39

3.4.1 STE Discordant Results for EPA Classification in a Bottom-Up Approach .......................................................................................................... 40

4.0 STE Test Method Reliability .................................................................................................... 43 5.0 Peer Review Summary .............................................................................................................. 44 6.0 References .................................................................................................................................. 46

Supplements

Supplements containing files all of the available documentation related to the short time exposure (STE) test method review have been provided on the enclosed CD-ROM. These documents are also available on the NICEATM website at http://ntp.niehs.nih.gov/go/41456. Supplement A contains the Kao Corporation STE background review document (A1) and appendices (A2 to A10) as submitted. Supplement B (B1 to B3) contains the data and information used by NICEATM to conduct a technical review of the STE test method.

Supplement A Kao Corporation Background Review Document and Appendices

Supplement A1 Kao Background Review Document: Current Status of In Vitro Test Methods for Identifying Ocular Irritants: Short Time Exposure (STE) Test

Supplement A2 Short Time Exposure (STE) Test Protocol


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Supplement A3 Chemical Classes of Substances Tested in the STE Test (Appendix B1) In Vivo Data Source of Substances Tested in the STE Test (Appendix B2) Solubility of Substances Tested in the STE Test (Appendix B3) Skin Corrosivity/Irritation of Substances Tested in the STE Test (Appendix B4) MTT Reduction (Appendix B5) of Substances Tested in the STE Test

Supplement A4 In Vitro Data for Substances Tested in the STE Test Sorted by Reference (Appendix C1) In Vitro Data for Substances Tested in the STE Test Sorted by Substance Name (Appendix C2)

Supplement A5 Comparison of In Vivo and In Vitro Ocular Irritancy Classifications Sorted by Reference (Appendix D1) Comparison of In Vivo and In Vitro Ocular Irritancy Classifications Sorted by Substance Name (Appendix D2)

Supplement A6 Intralaboratory CV Analysis of STE by Study

Supplement A7 In Vitro Data for Substances Tested in the STE Test: Sorted by Reference (Appendix F1) In Vitro Data for Substances Tested in the STE Test: Sorted by Substance Name with 0.05% Data (Appendix F2)

Supplement A8 Comparison of In Vivo and In Vitro Ocular Irritancy Classifications Sorted by Reference (Appendix G1) Comparison of In Vivo and In Vitro Ocular Irritancy Classifications Sorted by Substance Name with 0.05% Data (Appendix G2)

Supplement A9 EpiOcular Assay Protocol (Appendix H)

Supplement A10 In Vitro Data for Substances Tested in the EpiOcular Test

Supplement B Comparison of In Vitro and In Vivo Ocular Irritancy Classification

Supplement B1 In Vivo Classification

Supplement B2 STE Data Sorted by Study

Supplement B3 STE Data Sorted by Substance with Consensus Classifications


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List of Tables

Table 1 Overall STE Performance in a Top-Down Approach ........................................................ 2

Table 2 Top-Down Performance of Validated In Vitro Test Methods Compared to the STE Test Method ....................................................................................................................... 2

Table 3 Overall STE Performance in a Bottom-Up Approach ....................................................... 2

Table 4 Overall STE Performance in a Bottom-Up Approach When Substances with Vapor Pressure ≥6kPa and Nonsurfactant Solids are Excluded ................................................... 3

Table 5 Bottom-Up Performance of Validated In Vitro Test Methods Compared to the STE Test Method ....................................................................................................................... 3

Table 2-1 Test Substances Used to Evaluate STE Performance in a Top-Down Approacha ............. 8

Table 2-2 Test Substances Used to Evaluate STE Performance in a Bottom-Up Approacha .......... 15

Table 2-3 Test Substances Used to Evaluate STE Performance in a Bottom-Up Approach Within the Defined Applicability Domaina,b ................................................................... 22

Table 3-1 STE Performance for GHS Classification in a Top-Down Approach ............................. 31

Table 3-2 STE False Positive and False Negative Rates by Chemical Category and Properties of Interest for GHS Classification in a Top-Down Approach ......................................... 32

Table 3-3 STE Performance for GHS Classification in a Top-Down Approach After Excluding Discordant Categories .................................................................................... 33

Table 3-4 STE Performance for GHS Classification in a Bottom-Up Approach ............................ 34

Table 3-5 STE False Positive and False Negative Rates by Chemical Category and Properties of Interest for GHS Classification in a Bottom-Up Approach......................................... 35

Table 3-6 STE Performance for GHS Classification in a Bottom-Up Approach After Excluding Discordant Categories .................................................................................... 36

Table 3-7 STE Performance for EPA Classification in a Top-Down Approach .............................. 37

Table 3-8 STE False Positive and False Negative Rates by Chemical Category and Properties of Interest for EPA Classification in a Top-Down Approach .......................................... 38

Table 3-9 STE Performance for EPA Classification in a Top-Down Approach After Excluding Discordant Categories .................................................................................... 39

Table 3-10 STE Performance for EPA Classification in a Bottom-Up Approach ............................. 39

Table 3-11 STE False Positive and False Negative Rates by Chemical Category and Properties of Interest for EPA Classification in a Bottom-Up Approach ......................................... 40

Table 3-12 STE Performance for EPA Classification in a Bottom-Up Approach After Excluding Discordant Categories .................................................................................... 42


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List of Abbreviations and Acronyms

AD Applicability domain BCOP Bovine corneal opacity and permeability BRD Background review document CASRN CAS Registry Number® (American Chemical Society) CV Coefficient of variation EPA U.S. Environmental Protection Agency GHS Globally Harmonized System of Classification and Labelling of Chemicals ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods ICE Isolated chicken eye ILS Integrated Laboratory Systems, Inc. JaCVAM Japanese Center for the Validation of Alternative Methods kPa Kilopascals MeSH® Medical Subject Headings (National Library of Medicine) MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NICEATM National Toxicology Program (NTP) Interagency Center for the Evaluation of

Alternative Toxicological Methods NIEHS National Institute of Environmental Health Sciences NLM National Library of Medicine NTP National Toxicology Program OECD Organisation for Economic Co-operation and Development SIRC Statens Seruminstitut rabbit cornea STE Short time exposure TG Test Guideline UN United Nations Trademarks are the property of their respective owners.


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Acknowledgements

National Institute of Environmental Health Science

National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)

Warren Casey, PhD, DABT Acting Director, Project Officer

NICEATM Support Contract Staff (Integrated Laboratory Systems [ILS], Inc.)

David Allen, PhD (through 2011) Lori Rinckel, PhD Jonathan Hamm, PhD Elizabeth Lipscomb, PhD (through 2012) Steven Morefield, MD Catherine Sprankle James Truax, MA Linda Wilson


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Executive Summary

The short time exposure (STE) test method is an in vitro method for identifying ocular irritants. Developed by Takahashi et al. (2008), the STE test method assesses cytotoxicity in a rabbit corneal epithelial cell line (SIRC cells) through a 5-minute exposure to the test substance. In March 2011, Kao Corporation (Tochigi, Japan) submitted a background review document (BRD) titled “Current Status of In Vitro Test Methods for Identifying Ocular Irritants: Short Time Exposure (STE) Test” to the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). NICEATM conducted a preliminary evaluation of the BRD and requested additional information, which resulted in several revisions to the BRD. Kao Corporation drafted a final BRD in May 2012 (Supplement A). The BRD contains all data and information that were available in the peer-reviewed literature and Kao Corporation in–house data to describe the current validation status of the STE test method, including what was known about its accuracy and reliability.

This summary review document presents an evaluation of STE test method accuracy, sensitivity, specificity, false positive rate, and false negative rate based on test substances with corresponding in vivo data. The analysis in a top-down and a bottom-up approach was based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2011) and U.S. Environmental Protection Agency (EPA 2012) classification systems for eye hazard classification and labeling.

In a top-down approach, the STE test method is used to distinguish and label severe eye irritants/corrosives from all other hazard categories. Any substance not identified as a severe eye irritant/corrosive by the STE test method requires additional testing with other methods. A top-down approach requires a low false positive rate to avoid overclassification of substances. The false negative rate is not as critical because substances that test negative in the STE test method would be tested with another method.

In contrast, a bottom-up approach is used to distinguish substances not labeled as eye irritants from all other hazard categories. Any substance that tests positive in a bottom-up approach requires additional testing with other methods to determine the appropriate hazard classification and labeling. A bottom-up approach requires a low false negative rate to avoid irritants being classified and mislabeled as irritants when the correct eye hazard classification is GHS Not classified or EPA Category IV (minimal effects clearing in less than 24 hours). The false positive rate is not as critical because substances that test positive in the STE test method would be tested with another method.

The Kao Corporation BRD describes their analyses of 119 tests substances in four studies (Kojima et al. [Kao BRD]; Sakaguchi et al. 2011; Takahashi et al. 2009, 2010), with additional in-house data provided by Kao Corporation. In September 2012, Kao Corporation provided data on 52 additional surfactant or surfactant-containing substances, for a total of 169 substances with in vitro STE and in vivo rabbit eye test data (Supplement B).

The analyses in this report used consensus calls for both STE and rabbit eye test data when results were available from more than one laboratory or study. When equivocal results were obtained in two or more laboratories or in different studies, the more severe hazard classification was used.

Table 1 summarizes overall performance of the STE test method in a top-down approach for all substances in the database. The overall false positive rate in a top-down approach ranged from 1.2% (1/84) for GHS classification to 2.3% (2/87) for EPA classification. Exclusion of alcohols reduced the rate to 0% (data not shown). The performance of the STE test method is compared to other validated in vitro test methods in Table 2.


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Table 1 Overall STE Performance in a Top-Down Approach

Regulatory System N

Accuracy Sensitivity Specificity False

Positive Rate

False Negative Rate

% No. % No. % No. % No. % No. GHS 120 85 102/120 53 19/36 99 83/84 1.2 1/84 47 17/36 EPA 120 87 104/120 58 19/33 98 85/87 2.3 2/87 42 14/33

Abbreviations: EPA = U.S. Environmental Protection Agency; GHS = Globally Harmonized System of Classification and Labelling of Chemicals; N = number of substances; STE = short time exposure.

Table 2 Top-Down Performance of Validated In Vitro Test Methods Compared to the STE Test Method

GHS N Accuracy False Positive Rate False Negative Rate

% No. % No. % No. BCOP 188 79 149/188 24 29/123 15 10/65

ICE 144 83 120/144 8.0 9/114 50 15/30 CM 82 90 74/82 2.0 1/48 21 7/34 STE 120 85 102/120 1.2 1/84 47 17/36

Abbreviations: BCOP = bovine corneal opacity and permeability; CM = Cytosensor microphysiometer; GHS = Globally Harmonized System of Classification and Labelling of Chemicals; ICE = isolated chicken eye; N = number of substances.

The STE overall performance in a bottom-up approach is shown in Table 3. The overall false negative rate in a bottom-up approach ranged from 12.3% (9/73) for GHS classification to 24.7% (24/97) for EPA classification.

Table 3 Overall STE Performance in a Bottom-Up Approach

Regulatory System N

Accuracy Sensitivity Specificity False Positive Rate

False Negative Rate

% No. % No. % No. % No. % No. GHS 129 85 109/129 88 64/73 80 45/56 20 11/56 12 9/73 EPA 129 80 103/129 75 73/97 94 30/32 6.3 2/32 25 24/97

Abbreviations: EPA = U.S. Environmental Protection Agency; GHS = Globally Harmonized System of Classification and Labelling of Chemicals; N = number of substances; STE = short time exposure.

The applicability domain was evaluated to reduce the false positive rate and increase performance for both GHS and EPA classifications in a bottom-up approach. Improvements in the applicability domain were determined by analyzing assay performance by chemical class and physical properties. As a result, two applicability domains were evaluated based on excluding certain chemical and product classes, or physical characteristics. Applicability domain one excludes liquids with vapor pressures ≥6 kilopascals (kPa) solid alcohols, hydrocarbons, and salts while applicability domain two excludes liquids with vapor pressures ≥6 kilopascals (kPa) and nonsurfactant solids (Table 4). The performance of the STE test method is compared to other validated in vitro test methods in Table 5.


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Table 4 Overall STE Performance in a Bottom-Up Approach After Excluding Discordant Categories

Regulatory System N

Accuracy False Positive Rate False Negative Rate % No. % No. % No.

Applicability Domain 1 - Exclusion of liquids with vapor pressures ≥6 kilopascals (kPa) solid alcohols, hydrocarbons, and salts GHS 94 90 85/94 18 8/45 2 1/49 EPA 94 83 78/94 7.7 2/26 21 14/68

Applicability Domain 2 - Exclusion of liquids with vapor pressures ≥6 kilopascals (kPa) and nonsurfactant solids

GHS 101 90 91/101 19 9/47 1.9 1/54 EPA 101 85 86/101 7.1 2/28 18 13/73

Abbreviations: EPA = U.S. Environmental Protection Agency; GHS = Globally Harmonized System of Classification and Labelling of Chemicals; No. = number; STE = short time exposure.

Table 5 Bottom-Up Performance of Validated In Vitro Test Methods Compared to the STE Test Method

GHS N Accuracy False Positive Rate False Negative Rate

% No. % No. % No.

BCOP 188 66 125/188 69 63/91 0 0/97 CM 53 68 36/53 68 17/25 0 0/28

STE – AD1 94 91 85/94 18 8/45 2 1/49 STE – AD2 101 90 91/101 19 9/47 1.9 1/54 Abbreviations: AD = applicability domain; BCOP = bovine corneal opacity and permeability; CM = Cytosensor

microphysiometer; N = number of substances; STE = short time exposure. This evaluation of the STE performance shows that this method is able to distinguish substances as severe irritants or corrosives (i.e., GHS Category 1 or EPA Category I) from all other hazard categories (GHS Category 2A, 2B, Not Classified or EPA Category II, III, IV) in a top-down approach, with false positive rates ranging from 1.2% (1/84) to 2.3% (2/87) for the GHS and EPA classification systems, respectively. Exclusion of discordant chemical classes (e.g., alcohols, ethers, hydrocarbons, or nonionic surfactants) reduced the false positive rate to 0%. In a bottom-up approach to distinguish substances that were either not classified or minimal irritants (i.e., GHS Not Classified or EPA Category IV) from all other hazard categories (i.e., GHS Category 1, 2A, 2B or EPA Category I, II, III), the STE false negative rates ranged from 12.3% (9/73) to 24.7% (24/97). The range of false negative rates in a bottom-up approach was decreased to 2% (1/49) and 21% (14/68) for the GHS and EPA classification systems, respectively, when liquids with vapor pressures >6kPa solid alcohols, hydrocarbons, and salts were excluded. The range of false negative rates in a bottom-up approach was decreased to 1.9% (1/54) and 18% (13/73) for the GHS and EPA classification systems, respectively, when liquids with vapor pressures >6kPa and nonsurfactant solids were excluded.

This SRD along with the original Kao BRD and other supporting documentation were forward by NTP to four external scientific reviewers. The reviewers were provided a list of questions that included a request to comment on the adequacy of the database used for evaluating STE, the adequacy of the performance evaluation, and to provide comments for regulators using the test


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method. In response, the reviewers indicated that the database of compounds was generally sufficient and the review thorough. A summary of reviewer comments is provided in Section 5.0.


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1.0 Introduction and Background The Draize rabbit eye test has been the primary method used to determine the ocular irritation potential of chemicals (Draize et al. 1944). However, public interest in animal welfare has increased the pressure to develop non-animal alternatives. The development of alternative methods is also accelerating due to regulations banning animal ocular irritation tests for cosmetics in the European Union (Directive 2003/15/EC; European Union 2003). As a result, numerous alternative ocular irritation methods that use cell lines and tissues are being developed around the world (Balls et al. 1999; Eskes et al. 2005; Ohno et al. 1999). The test guidelines for the bovine corneal opacity and permeability (BCOP) test method and isolated chicken eye (ICE) test method were accepted by the Organisation for Economic Co-operation and Development (OECD) for predicting severe ocular irritation (OECD 2012a, 2012b). However, no other test guidelines have been accepted for in vitro ocular irritation tests.

The short time exposure (STE) test method is an alternative ocular irritation method developed by Kao Corporation (Takahashi et al. 2008). The STE test method uses a cultured cell line (SIRC cells) derived from rabbit cornea and uses shorter exposure times than many other cytotoxicity-based methods. Generally, cytotoxicity tests using cultured cells have the advantage of being simple, quick procedures with a low evaluation cost. The facility requirements necessary to conduct the STE test include a standard laboratory setup for cell culture. The cornea is one of the main targets during accidental eye exposures, and damage to the cornea can result in visual impairment. A final advantage of the STE test method is that it can be used to evaluate poorly water-soluble chemicals like toluene, octanol, and hexanol by using mineral oil as the vehicle (Takahashi et al. 2008).

The STE test method involves exposing SIRC cells to 5% and 0.05% concentrations of test substance for 5 minutes. Following exposure to 5% test substance concentration, substances that reduce cell viability below 70% are classified as irritants. Using this classification scheme, Kao Corporation assessed the performance of the STE test method in a bottom-up approach to distinguish substances not labeled as irritants from all other categories. Kao Corporation also proposed a second approach to establish an ocular irritation potency ranking that differentiates severely irritating substances from mild and moderate irritants. This approach uses a point system based on the test concentration and relative viability resulting from an exposure to 5% or 0.05% of test material (Takahashi et al. 2008). This second approach was used to review the STE test method in a top-down approach to distinguish corrosives/severe irritants from all other categories.

In March 2011, the Japanese Center for the Validation of Alternative Methods (JaCVAM), as part of the International Cooperation on Alternative Test Methods agreement, requested that the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) conduct a technical review of the STE test method. In support of the review, Kao Corporation submitted their STE test method BRD and subsequently provided NICEATM with a revised May 2012 BRD (Supplement A). The BRD contains STE and rabbit eye test data for 119 substances from four in vitro–in vivo comparative studies, with additional in-house data on 23 substances provided by Kao Corporation. After the preliminary analysis, additional data were requested and provided for 52 surfactants or surfactant-containing formulations that increased the STE database to 169 substances.

To assess the ability of the STE test method to predict the regulatory hazard classification identified in the rabbit eye test, the STE rank results were converted to Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and U.S. Environmental Protection Agency (EPA) classifications (UN 2011, EPA 2012). An STE rank of 1 (nonirritant), 2 (mild or moderate irritant), or 3 (severe irritant) was converted to GHS Not Classified, Category 2A/2B, or Category 1, respectively,


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or to the U.S. EPA Label Review Manual classification of eye irritation as Category IV, Category III/II, or Category I, respectively.

STE test method performance was also evaluated in a top-down approach (i.e., distinguishing GHS Category 1 or EPA Category I substances from those in all other categories) or in a bottom-up approach (i.e., distinguishing GHS Not Classified or EPA Category IV substances from all other categories) for substances with corresponding in vitro–in vivo data. For a top-down approach, 120 substances had corresponding in vitro and in vivo classification data using the GHS or EPA classification systems, respectively, that were suitable for accuracy analysis. For a bottom-up approach, 129 substances had suitable in vitro–in vivo data for GHS and EPA classifications.

A variety of chemical categories were tested in the STE test method, and the chemical categories with the greatest amount of test data are alcohols, carboxylic acids, esters, ethers/polyethers, heterocyclic compounds, ketones/lactones, onium compounds, and salts. Physical properties of these substances have also been evaluated (pH, solids, liquids, and surfactants [nonionic, anionic, cationic]). This summary review document describes evaluations of the STE test method performance in a top-down or bottom-up approach.


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2.0 STE Test Method Database The May 2012 BRD submitted by Kao Corporation (Supplement A) includes information on the STE test method, the test method protocol, and data and performance analyses based on:

• Takahashi et al. (2009) – Prevalidation study 1 on 44 substances • Takahashi et al. (2010) – Prevalidation study 2 on 70 substances • Sakaguchi et al. (2011) – Phase I validation study on 25 substances • Kojima et al. (Kao BRD) – Phase II validation study on 40 substances, then combined with Phase

I substances for a total of 63 substances • Kao in-house data on 22 of 23 substances in the original BRD

Additional information on the test substances are found in Supplement B. Supplement B1 contains the in vivo data used to develop consensus in vivo classifications for substances evaluated in the STE test method. Supplement B2 shows the test substances along with CAS Registry Number® (American Chemical Society), concentration tested, STE test data (mean viability value, standard deviation, number of replicates), category classification, and the reference. Supplement B3 provides the same information but indicates the consensus STE classification.

The STE database includes test substances in the Kao Corporation BRD, with additional data on 52 surfactants and surfactant-containing formulations provided by Kao Corporation. However, the database used to assess performance consists of consensus classifications when a single substance was tested in multiple laboratories or in different studies. In vivo data are typically generated by testing neat chemicals. Twenty-three substances that were tested in the STE test method at a concentration less than 100% and that did not produce a severe irritant effect were excluded from these analyses because a mild/moderate irritant or nonirritant classification of a diluted chemical may be classified as a severe irritant when tested neat in vitro.

Chemicals that directly reduce MTT in the absence of cells have been shown to artificially inflate viability measures and underpredict cytotoxicity (Huang 2004; Sims and Plattner 2009). Kao Corporation assessed chemicals for their ability to directly reduce MTT by incubating the test substances with MTT and visually inspecting for color development. Test substances that were identified as direct MTT reducers were removed from top-down analysis and those classified as STE nonirritants were removed from bottom-up analysis, as these could be false negative.

Finally, in vivo data were analyzed to calculate the appropriate ocular irritation hazard classification. These data include cornea, iris, and conjunctiva scores for each animal at 24, 48, and 72 hours following test substance administration and/or assessment of lesions at 7, 14, and 21 days. Some test substances had insufficient in vivo data to assign a hazard classification. Thus, these substances were not used to evaluate STE accuracy and reliability.

The STE database contains 169 test substances representing a variety of chemical classes and physicochemical properties. Table 2-1 provides information on the test substances evaluated in a top-down approach to identify severe eye irritants or corrosives. Table 2-2 provides information evaluated in a bottom-up approach to identify GHS Not Classified or EPA Category IV substances. These substances had corresponding in vivo data, were assigned a GHS (UN 2011) or EPA (EPA 2012) eye hazard classification, and met other assay criteria as discussed in Section 3.2. Table 2-3 shows the substances used to assess the STE test method in a bottom-up approach applicability domain one excluding liquids with vapor pressure >6 kilopascals alcohols, hydrocarbons, and salts and applicability domain two excluding liquids with vapor pressure >6 kilopascals and nonsurfactant solids.


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Table 2-1 Test Substances Used to Evaluate STE Performance in a Top-Down Approacha

Test Substance CASRN Physical Form as Tested

Vapor Pressure

(kPa 25°C)b

In Vivo Conc (%)c

GHS Consensusd

EPA Consensusd

Overall Consensus STE Ranke

1-Bromo-4-chlorobutane 6940-78-9 Liquid 0.164 100% Not Classified Category IV 1

1-Dodecanaminium, N-(2-hydroxy-3-sulfopropyl)-N,N-dimethyl-, inner salt

13197-76-7 Liquid 1.16E-21 100% Category 1 Category I 2

1-Methylpropyl benzene 135-98-8 Liquid 0.176 100% Not Classified Category IV 1

1-Octanol 111-87-5 Liquid 0.013 100% Category 2A Category II 2

1,3-Di-isopropylbenzene 99-62-7 Liquid 0.041 100% Not Classified Category IV 1

1,5-Hexadiene 592-42-7 Liquid 28.6 100% Not Classified Category III 1

1,9-Decadiene 1647-16-1 Liquid 0.320 100% Not Classified Category IV 1

2-Benzyloxyethanol 622-08-2 Liquid 2.9E-4 100% Category 2A Category II 2

2-Ethoxyethyl acetate (Cellosolve acetate) 111-15-9 Liquid 0.397 100% Not Classified Category III 1

2-Ethyl-1-hexanol 104-76-7 Liquid 0.025 100% Category 2A Category II 2

2-Ethylhexyl p-dimethylamino benzoate 21245-02-3 Liquid 4.72E-06 100% Not Classified Category IV 1

2-Methyl-1-pentanol 105-30-6 Liquid 0.191 100% Category 2B Category III 2

2-Methylbutyric acid 116-53-0 Liquid 0.149 100% Category 1 Category I 2

2-Methylpentane 107-83-5 Liquid 27.8 100% Not Classified Category IV 1

2-Naphthalenesulfonic acid,6-hydroxy-,monosodium salt, polymer with formaldehyde and hydroxymethylbenzenesulfonic aid monosodium salt

85255-76-1 Liquid NA 100% Category 1 Category II 2

2,2-Dimethyl-3-pentanol 3970-62-5 Liquid 0.413 100% Not Classified Category III 1


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Vapor Pressure

(kPa 25°C)b

In Vivo Conc (%)c

GHS Consensusd

EPA Consensusd


2,4-Pentanediol 625-69-4 Liquid 7.3E-03 100% Not Classified Category IV 1

2,5-Dimethyl-2,5-hexanediol 110-03-2 Solid 5.78E-04 100% Category 1 Category I 1

3-Methoxy-1,2-propanediol 623-39-2 Liquid 1.92E-03 100% Not Classified Category IV 1

3-Methylhexane 589-34-4 Liquid 8.29 100% Not Classified Category IV 1

3,3-Dimethylpentane 562-49-2 Liquid 10.1 100% Not Classified Category IV 1

Acetone 67-64-1 Liquid 33.2 100% Category 2A Category II 2

Acid red 92 18472-87-2 Solid 5.71E-24 100% Category 1 Category I 3

Acrylic acid homopolymer sodium salt 9003-04-7 Solid 4.56E-04 100% Not Classified Category IV 1

Ammonium nitrate 6484-52-2 Solid 4.48E-16 100% Category 2B Category III 1

Benzalkonium chloride 8001-54-5 Liquid NA 100% Category 1 Category I 3

Benzalkonium chloride (10%) 63449-41-2 Solid NA 10% Category 1 Category I 3

Benzene, 1,1'-oxybis-, tetrapropylene derivatives, sulfonated, sodium salts

119345-04-9 Solid NA 100% Category 1 Category I 3

Benzyl alcohol 100-51-6 Liquid 7.14E-03 100% Category 1 Category I 2

Body shampoo A NA Liquid NA 100% Category 2A Category II 2

Butanol 71-36-3 Liquid 1.04 100% Category 1 Category I 2

Butyl acetate 123-86-4 Liquid 1.59 100% Not Classified Category III 1

Butyl cellosolve 111-76-2 Liquid 0.0633 100% Category 1 Category II 2

Butylnaphthalenesulfonic acid sodium salt 25638-17-9 Solid NA 100% Category 1 Category I 2

Butyrolactone 96-48-0 Liquid 0.0394 100% Category 2A Category II 2


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Vapor Pressure

(kPa 25°C)b

In Vivo Conc (%)c

GHS Consensusd

EPA Consensusd


Camphene 79-92-5 Solid 0.237 100% Category 2B Category III 1

Cetylpyridinium bromide (10%) 140-72-7 Solid 3.47E-07 10% Category 1 Category I 3

Cetylpyridinium chloride 6004-24-6 Solid NA 10% Category 1 Category I 3

Cetyltrimethylammonium bromide (10%) 57-09-0 Solid NA 10% Category 1 Category I 3

Cyclohexanol 108-93-0 Liquid 0.087 100% Category 1 Category I 2

Cyclohexanone 108-94-1 Liquid 0.539 100% Not Classified Category III 2

Cyclopentanol 96-41-3 Liquid 0.307 100% Category 2B Category II 2

Di-n-propyl disulphide 629-19-6 Liquid 0.0664 100% Not Classified Category IV 1

Di(2-Ethylhexyl) sodium sulfosuccinate 577-11-7 Solid 1.63E-15 10% Category 1 Category I 3

Di(propylene glycol) propyl ether 29911-27-1 Liquid 2.38E-04 100% Category 2B Category III 2

Diisobutyl ketone 108-83-8 Liquid 0.287 100% Not Classified Category IV 1

Dimethyl sulfoxide 67-68-5 Liquid 0.0829 100% Not Classified Category III 1

Dodecane 112-40-3 Liquid 0.0315 100% Not Classified Category III 1

Domiphen bromide 538-71-6 Solid NA 10% Category 1 Category I 3

Ethanol 64-17-5 Liquid 812 100% Category 2A Category I 1

Ethyl 2-methylacetoacetate 609-14-3 Liquid 0.0915 100% Category 2B Category III 2

Ethyl acetate 141-78-6 Liquid 13.1 100% Not Classified Category III 2

Ethyl trimethyl acetate 3938-95-2 Liquid 2.24 100% Not Classified Category III 1

Ethylhexyl salicylate 118-60-5 Liquid 9.51E-07 100% Not Classified Category IV 1

Glycerol 56-81-5 Liquid 1.06E-05 100% Not Classified Category IV 1

Glycidyl methacrylate 106-91-2 Liquid 0.0829 100% Not Classified Category III 2


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Vapor Pressure

(kPa 25°C)b

In Vivo Conc (%)c

GHS Consensusd

EPA Consensusd


Imidazole 288-32-4 Solid 5.78E-04 100% Category 1 Category I 2

Iso-octyl acrylate 29590-42-9 Liquid 0.0204 100% Not Classified Category IV 1

Isobutanal 78-84-2 Liquid 21.9 100% Category 2B Category III 2

Isobutyl alcohol 78-83-1 Liquid 1.78 100% Category 1 Category I 2

Isopropyl alcohol 67-63-0 Liquid 6.61 100% Category 2A Category III 1

Isopropyl bromide 75-26-3 Liquid 28.5 100% Not Classified Category IV 1

Isopropyl myristate 110-27-0 Liquid 1.08E-04 100% Not Classified Category IV 1

Lactic acid 50-21-5 Liquid 3.81E-03 100% Category 1 Category I 2

Lauryldimethylamine oxide 1643-20-5 Solid 1.68E-15 100% Category 1 Category I 3

Lotion A NA Liquid NA 100% Not Classified Category IV 1

m-Phenylene diamine 108-45-2 Solid 251E-04 100% Category 1 Category I 2

Methoxyethyl acrylate 3121-61-7 Liquid 0.598 100% Category 1 ≥Category III 2

Methyl acetate 79-20-9 Liquid 7.03 100% Category 2A Category II 1

Methyl amyl ketone 110-43-0 Liquid 0.655 100% Not Classified Category III 1

Methyl cyanoacetate 105-34-0 Liquid 0.047 100% Category 2A Category II 2

Methyl cyclopentane 96-37-7 Liquid 17.8 100% Not classified Category III 1

Methyl ethyl ketone (2-Butanone) 78-93-3 Liquid 13.1 100% Category 2A Category III 2

Methyl isobutyl ketone 108-10-1 Liquid 2.90 100% Not Classified Category III 1

Methyl trimethyl acetate 598-98-1 Liquid 4.76 100% Not Classified Category IV 1

Myristyl alcohol 112-72-1 Solid 269E-05 100% Category 2A Category III 1

n-Hexanol 111-27-3 Liquid 0.117 100% Category 2A Category II 2

n-Hexyl bromide 111-25-1 Liquid 0.541 100% Not Classified Category IV 1


12


Vapor Pressure

(kPa 25°C)b

In Vivo Conc (%)c

GHS Consensusd

EPA Consensusd


n-Octyl bromide 111-83-1 Liquid 0.0691 100% Not Classified Category IV 1

n,n-Dimethylguanidine sulfate 598-65-2 Solid 4.04 100% Not Classified Category III 1

Naphthalenesulfonic acid, butyl-, polymer with formaldehyde and 2-naphthalenesulfonic acid, sodium salt

188070-49-7 Solid NA 100% Category 2A Category II 2

Polyethylene glycol 400 25322-68-3 Liquid NA 100% Not Classified Category IV 1

Polyethyleneglycol monolaurate (10 E.O.) 9004-81-3 Liquid 0 100% Not Classified Category IV 2

Polyoxyethylene hydrogenated castor oil (60E.O.)

61788-85-0 Solid NA 100% Not Classified Category IV 1

Polyoxyethylene(10) polyoxypropylene(1.5) lauryl-myristyl ether

68439-51-0 Liquid NA 100% Category 1 Category I 3

Polyoxyethylene(13) (mono-, di-, tri-) styrenated phenyl ether

104376-75-2 Liquid NA 100% Not Classified Category III 3

Polyoxyethylene(14) tribenzylated phenyl ether 116998-28-8 Liquid NA 100% Not Classified Category IV 1

Polyoxyethylene(160) sorbitan triisostearate 54392-28-8 Solid NA 100% Not Classified Category IV 1


104376-75-2 Liquid NA 100% Not Classified Category II 2

Polyoxyethylene(23) lauryl ether 9002-92-0 Solid 2.03E-13 100% Category 2A Category III 2


13


Vapor Pressure

(kPa 25°C)b

In Vivo Conc (%)c

GHS Consensusd

EPA Consensusd


Polyoxyethylene(40) hydrogenated castor oil 61788-85-0 Liquid NA 100% Not Classified Category IV 1

Potassium laurate 10124-65-9 Solid 0 10% Category 1 Category I 3

Potassium oleate 143-18-0 Solid 4.93E-10 100% Not Classified Category III 2

Promethazine hydrochloride 58-33-3 Solid 0 100% Category 1 Category I 3

Propasol solvent P 1569-01-3 Liquid 0.180 100% Category 2B Category II 2

Propylene glycol 57-55-6 Liquid 0.0148 100% Not Classified Category IV 1

Pyridine 110-86-1 Liquid 2.58 100% Category 1 Category I 2

Rinse A NA Liquid NA 100% Not Classified Category III 2

Rinse B NA Liquid NA 100% Category 2B Category III 2

Rinse C NA Liquid NA 100% Not Classified Category IV 1

Rinse D NA Liquid NA 100% Not Classified Category III 1

Shampoo A NA Liquid NA 100% Category 2A Category II 2

Shampoo B NA Liquid NA 100% Category 1 Category I 2

Shampoo C NA Liquid NA 100% Category 2A Category II 2

Shampoo D NA Liquid NA 100% Category 2A Category II 2

Sodium 2-naphthalenesulfonate 532-02-5 Solid NA 100% Not Classified Category III 2

Sodium hydroxide 1310-73-2 Solid 6.53E-21 10% Category 1 Category I 3

Sodium lauryl sulfate 151-21-3 Solid 2.40E-13 100% ≥Category 2A Category III 3

Sodium lauryl sulfate (15%) 151-21-3 Solid NA 15% Category 1 Category I 3

Sodium monochloroacetate 3926-62-3 Solid 4.23E-09 100% Category 2B Category III 2


14


Vapor Pressure

(kPa 25°C)b

In Vivo Conc (%)c

GHS Consensusd

EPA Consensusd


Sodium polyoxyethylene(3) lauryl ether sulfate


Sodium salicylate 54-21-7 Solid 4.84E-12 100% Category 1 Category I 1

Sorbitan monolaurate 1338-39-2 Liquid 1.25E-15 100% Not Classified Category IV 2

Stearyltrimethylammonium chloride 112-03-8 Solid NA 10% Category 1 Category I 3

Styrene 100-42-5 Liquid 0.673 100% Not Classified Category III 1

Toluene 108-88-3 Liquid 3.16 100% ≥Category 2B Category III 1

Triethanolamine 102-71-6 Liquid 4.51E-07 100% Not Classified Category III 1

Triethanolamine polyoxyethylene(3.0) lauryl ether sulfate


Triton X-100 9002-93-1 Liquid 0 100% Category 1 Category I 3

Triton X-100 (10%) 9002-93-1 Liquid 9.32E-04 10% Category 1 Category I 2

Tween 20 9005-64-5 Liquid 0 100% Not Classified Category III 2

Tween 80 9005-65-6 Liquid 0 100% Not Classified Category IV 1 Abbreviations: CASRN = CAS Registry Number® (American Chemical Society); EPA = U.S. Environmental Protection Agency; GHS = United Nations Globally Harmonized

System of Classification and Labelling of Chemicals; JaCVAM = Japanese Center for the Validation of Alternative Methods; kPa = kilopascals; NA = not available; STE = short time exposure.

a A top-down approach is used to distinguish severe eye irritants or corrosives (i.e., GHS Category 1, EPA Category I, or STE Rank 3) from all other hazard or no hazard categories (i.e., GHS Category 2A, 2B, Not Classified; EPA Category II, III, IV; or STE Rank 1 or 2).

b Vapor pressure is expressed in kilopascals at 25°C. Vapor pressures were found using the Hazardous Substances Data Bank (HSDB® [U.S. National Library of Medicine]), available at http://toxnet.nlm.nih.gov (accessed 2/25/2013) or from ChemSpider (available at www.chemspider.com [accessed 2/25/2013]). If actual values were not available, predicted values were obtained from the U.S. EPA EPI (Estimation Programs Interface) Suite™ for Microsoft® Windows, v. 4.11) or ACD/Labs’ ACD/PhysChem Suite available at http://www.acdlabs.com/products/pc_admet/physchem/physchemsuite/ (accessed 2/25/2013). Data from the EPI Suite and ACD/PhysChem Suite programs were also available in ChemSpider.

c The concentration as tested in the rabbit eye test, based on NICEATM data. For substances tested at 100%, the starting material was tested neat/undiluted. d The consensus classification of two or more studies. When there was no consensus using either the GHS (UN 2011) or EPA (EPA 2012) eye hazard classification system (e.g.,

one GHS Category 2A and one GHS Category 2B), the more hazardous classification (i.e., GHS Category 2A) was used as the consensus classification.

http://toxnet.nlm.nih.gov/


15

e STE rank scores from Kao Corporation were equated to the GHS or EPA classification of eye hazard (i.e., UN 2011 and EPA 2012) such that an STE rank of 3 was considered a severe eye irritant or corrosive (i.e., GHS Category 1 or EPA Category I); an STE rank of 2 was considered a moderate to mild eye irritant (i.e., GHS Category 2A or 2B or EPA Category II or III); and an STE rank of 1 was considered to be equivalent to GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours).

Table 2-2 Test Substances Used to Evaluate STE Performance in a Bottom-Up Approacha


Vapor Pressure

(kPa 25°C)b

In Vivo Conc

(%)c GHS

Consensusd EPA

Consensusd


1-Bromo-4-chlorobutane 6940-78-9 Liquid 0.164 100% Not Classified Category IV 1 1-Dodecanaminium, N-(2-hydroxy-3-sulfopropyl)-N,N-dimethyl-, inner salt


1-Methylpropyl benzene 135-98-8 Liquid 0.176 100% Not Classified Category IV 1 1-Octanol 111-87-5 Liquid 0.0132 100% Category 2A Category II 2 1,3-Di-isopropylbenzene 99-62-7 Liquid 0.041 100% Not Classified Category IV 1 1,5-Hexadiene 592-42-7 Liquid 28.6 100% Not Classified Category III 1 1,9-Decadiene 1647-16-1 Liquid 0.320 100% Not Classified Category IV 1 2-Benzyloxyethanol 622-08-2 Liquid 294E-04 100% Category 2A Category II 2 2-Ethoxyethyl acetate (Cellosolve acetate) 111-15-9 Liquid 0.397 100% Not Classified Category III 1

2-Ethyl-1-hexanol 104-76-7 Liquid 0.245 100% Category 2A Category II 2 2-Ethylhexyl p-dimethylamino benzoate 21245-02-3 Liquid 4.72E-06 100% Not Classified Category IV 1

2-Methyl-1-pentanol 105-30-6 Liquid 0.191 100% Category 2B Category III 2 2-Methylbutyric acid 116-53-0 Liquid 0.149 100% Category 1 Category I 2 2-Methylpentane 107-83-5 Liquid 27.8 100% Not Classified Category IV 1 2-Naphthalenesulfonic acid,6-hydroxy-,monosodium salt, polymer with formaldehyde and hydroxymethylbenzenesulfonic aid monosodium salt

85255-76-1 Liquid NA 100% Category 1 Category II 2


16


Vapor Pressure

(kPa 25°C)b

In Vivo Conc

(%)c GHS

Consensusd EPA

Consensusd


2,2-Dimethyl-3-pentanol 3970-62-5 Liquid 0.413 100% Not Classified Category III 1 2,4-Pentanediol 625-69-4 Liquid 7.3E-03 100% Not Classified Category IV 1 2,5-Dimethyl-2,5-hexanediol 110-03-2 Solid 578E-04 100% Category 1 Category I 1 3-Methoxy-1,2-propanediol 623-39-2 Liquid 1.92E-03 100% Not Classified Category IV 1 3-Methylhexane 589-34-4 Liquid 8.29 100% Not Classified Category IV 1 3,3-Dimethylpentane 562-49-2 Liquid 10.1 100% Not Classified Category IV 1 Acetic acid 64-19-7 Liquid 2.29 10% Category 1 Category I 2 Acetone 67-64-1 Liquid 33.2 100% Category 2A Category II 2 Acid red 92 18472-87-2 Solid 5.71E-24 100% Category 1 Category I 3 Acrylic acid homopolymer sodium salt 9003-04-7 Solid 4.56E-04 100% Not Classified Category IV 1

Ammonium nitrate 6484-52-2 Solid 4.48E-16 100% Category 2B Category III 1 Benzalkonium chloride 8001-54-5 Liquid NA 100% Category 1 Category I 3 Benzalkonium chloride (10%) 63449-41-2 Solid NA 10% Category 1 Category I 3

Benzene, 1,1'-oxybis-, tetrapropylene derivatives, sulfonated, sodium salts


Benzyl alcohol 100-51-6 Liquid 7.14E-03 100% Category 1 Category I 2 Body shampoo A NA Liquid NA 100% Category 2A Category II 2 Butanol 71-36-3 Liquid 1.04 100% Category 1 Category I 2 Butyl acetate 123-86-4 Liquid 1.595 100% Not Classified Category III 1 Butyl cellosolve 111-76-2 Liquid 0.0633 100% Category 1 Category II 2 Butylnaphthalenesulfonic acid sodium salt 25638-17-9 Solid NA 100% Category 1 Category I 2

Butyrolactone 96-48-0 Liquid 0.0394 100% Category 2A Category II 2 Calcium thioglycolate 5793-98-6 Solid 4.20E-03 100% Category 1 Category I 2


17


Vapor Pressure

(kPa 25°C)b

In Vivo Conc

(%)c GHS

Consensusd EPA

Consensusd


Camphene 79-92-5 Solid 0.237 100% Category 2B Category III 1 Cetyl trimethyl ammonium chloride 112-02-7 Liquid NA 5% Category 1 Category I 2

Cetylpyridinium bromide 140-72-7 Solid 3.47E-07 10% Category 1 Category I 3 Cetylpyridinium chloride 6004-24-6 Solid NA 10% Category 1 Category I 3 Cetyltrimethylammonium bromide 57-09-0 Solid NA 10% Category 1 Category I 3

Cetyltrimethylammonium bromide (10%) 57-09-0 Solid NA 10% Category 1 Category I 3

Cyclohexanol 108-93-0 Liquid 0.0866 100% Category 1 Category I 2 Cyclohexanone 108-94-1 Liquid 539 100% Not Classified Category III 2 Cyclopentanol 96-41-3 Liquid 307 100% Category 2B Category II 2 Di-n-propyl disulphide 629-19-6 Liquid 0.0664 100% Not Classified Category IV 1 Di(2-Ethylhexyl) sodium sulfosuccinate 577-11-7 Solid 1.63E-15 10% Category 1 Category I 3

Di(propylene glycol) propyl ether 29911-27-1 Liquid 2.38E-04 100% Category 2B Category III 2

Diethylethanolamine 100-37-8 Liquid 0.0863 100% Category 1 Category I 2 Diisobutyl ketone 108-83-8 Liquid 0.287 100% Not Classified Category IV 1 Dimethyl sulfoxide 67-68-5 Liquid 0.0829 100% Not Classified Category III 1 Distearyldimethylammonium chloride 107-64-2 Solid 2.55E-15 100% Category 1 Category I 2

Dodecane 112-40-3 Liquid 0.0315 100% Not Classified Category III 1 Domiphen bromide 538-71-6 Solid NA 10% Category 1 Category I 3 Ethanol 64-17-5 Liquid 8.12 100% Category 2A Category I 1 Ethyl 2-methylacetoacetate 609-14-3 Liquid 0.0915 100% Category 2B Category III 2 Ethyl acetate 141-78-6 Liquid 13.1 100% Not Classified Category III 2


18


Vapor Pressure

(kPa 25°C)b

In Vivo Conc

(%)c GHS

Consensusd EPA

Consensusd


Ethyl trimethyl acetate 3938-95-2 Liquid 2.240 100% Not Classified Category III 1 Ethylhexyl salicylate 118-60-5 Liquid 9.51E-07 100% Not Classified Category IV 1 Glycerol 56-81-5 Liquid 1.06E-05 100% Not Classified Category IV 1 Glycidyl methacrylate 106-91-2 Liquid 0.0829 100% Not Classified Category III 2 Imidazole 288-32-4 Solid 5.78E-04 100% Category 1 Category I 2 Iso-octyl acrylate 29590-42-9 Liquid 0.0204 100% Not Classified Category IV 1 Isobutanal 78-84-2 Liquid 21.9 100% Category 2B Category III 2 Isobutyl alcohol 78-83-1 Liquid 1.78 100% Category 1 Category I 2 Isopropyl alcohol 67-63-0 Liquid 6.61 100% Category 2A Category III 1 Isopropyl bromide 75-26-3 Liquid 28.5 100% Not Classified Category IV 1 Isopropyl myristate 110-27-0 Liquid .108E-04 100% Not Classified Category IV 1 Lactic acid 50-21-5 Liquid 3.81E-03 100% Category 1 Category I 2 Lauric acid 143-07-7 Solid 2.13E-09 100% ≥Category 2A ≥Category II 2 Lauryldimethylamine oxide 1643-20-5 Solid 1.68E-15 100% Category 1 Category I 3 Lotion A NA Liquid NA 100% Not Classified Category IV 1 m-Phenylene diamine 108-45-2 Solid 2.51E-04 100% Category 1 Category I 2 Methoxyethyl acrylate 3121-61-7 Liquid 0.598 100% Category 1 ≥Category III 2 Methyl acetate 79-20-9 Liquid 7.03 100% Category 2A Category II 1 Methyl amyl ketone 110-43-0 Liquid 0.655 100% Not Classified Category III 1 Methyl cyanoacetate 105-34-0 Liquid 0.0469 100% Category 2A Category II 2 Methyl cyclopentane 96-37-7 Liquid 17.8 100% Not Classified Category III 1 Methyl ethyl ketone (2-Butanone) 78-93-3 Liquid 13.1 100% Category 2A Category III 2

Methyl isobutyl ketone 108-10-1 Liquid 2.90 100% Not Classified Category III 1 Methyl trimethyl acetate 598-98-1 Liquid 4.76 100% Not Classified Category IV 1


19


Vapor Pressure

(kPa 25°C)b

In Vivo Conc

(%)c GHS

Consensusd EPA

Consensusd


Myristyl alcohol 112-72-1 Solid 2.69E-05 100% Category 2A Category III 1 n-Butanal 123-72-8 Liquid 14.4 100% Category 2B Category III 2 n-Hexanol 111-27-3 Liquid 0.117 100% Category 2A Category II 2 n-Hexyl bromide 111-25-1 Liquid 0.541 100% Not Classified Category IV 1 n-Octyl bromide 111-83-1 Liquid 0.0691 100% Not Classified Category IV 1 n,n-Dimethylguanidine sulfate 598-65-2 Solid 4.04 100% Not Classified Category III 1


188070-49-7 Solid NA 100% Category 2A Category II 2

Polyethylene glycol 400 25322-68-3 Liquid NA 100% Not Classified Category IV 1 Polyethyleneglycol monolaurate (10 E.O.) 9004-81-3 Liquid 0 100% Not Classified Category IV 2

Polyoxyethylene hydrogenated castor oil (60E.O.)

61788-85-0 Solid NA 100% Not Classified Category IV 1

Polyoxyethylene(10) polyoxypropylene(1.5) lauryl-myristyl ether

68439-51-0 Liquid NA 100% Category 1 Category I 3

Polyoxyethylene(13) (mono-, di-, tri-)styrenated phenyl ether

104376-75-2 Liquid NA 100% Not Classified Category III 3

Polyoxyethylene(14) tribenzylated phenyl ether 116998-28-8 Liquid NA 100% Not Classified Category IV 1

Polyoxyethylene(160) sorbitan triisostearate 54392-28-8 Solid NA 100% Not Classified Category IV 1


20


Vapor Pressure

(kPa 25°C)b

In Vivo Conc

(%)c GHS

Consensusd EPA

Consensusd



104376-75-2 Liquid NA 100% Not Classified Category II 2

Polyoxyethylene(20) hydrogenated tallow amine 61790-82-7 Solid NA 100% ≥Category 2A ≥Category II 3

Polyoxyethylene(23) lauryl ether 9002-92-0 Solid 2.03E-13 100% Category 2A Category III 2

Polyoxyethylene(40) hydrogenated castor oil 61788-85-0 Liquid NA 100% Not Classified Category IV 1

Potassium laurate 10124-65-9 Solid 0 10% Category 1 Category I 3 Potassium oleate 143-18-0 Solid 4.93E-10 100% Not Classified Category III 2 Promethazine hydrochloride 58-33-3 Solid 0 100% Category 1 Category I 3 Propasol solvent P 1569-01-3 Liquid 0.180 100% Category 2B Category II 2 Propylene glycol 57-55-6 Liquid 0.0148 100% Not Classified Category IV 1 Pyridine 110-86-1 Liquid 2.58 100% Category 1 Category I 2 Quaternary ammonium compounds, benzyl-C12-16-alkyldimethyl, chlorides


Quaternary ammonium compounds, di-C12-15-alkyldimethyl, chlorides


Rinse A NA Liquid NA 100% Not Classified Category III 2 Rinse B NA Liquid NA 100% Category 2B Category III 2 Rinse C NA Liquid NA 100% Not Classified Category IV 1 Rinse D NA Liquid NA 100% Not Classified Category III 1 Shampoo A NA Liquid NA 100% Category 2A Category II 2 Shampoo B NA Liquid NA 100% Category 1 Category I 2 Shampoo C NA Liquid NA 100% Category 2A Category II 2


21


Vapor Pressure

(kPa 25°C)b

In Vivo Conc

(%)c GHS

Consensusd EPA

Consensusd


Shampoo D NA Liquid NA 100% Category 2A Category II 2 Sodium 2-naphthalenesulfonate 532-02-5 Solid NA 100% Not Classified Category III 2

Sodium hydroxide 1310-73-2 Solid 6.53E-22 10% Category 1 Category I 3 Sodium lauryl sulfate 151-21-3 Solid 2.40E-13 100% ≥Category 2A Category III 3 Sodium lauryl sulfate (15%) 151-21-3 Solid NA 15% Category 1 Category I 3 Sodium monochloroacetate 3926-62-3 Solid 4.23E-09 100% Category 2B Category III 2 Sodium polyoxyethylene(3) lauryl ether sulfate 9004-82-4 Liquid 2.27E-13 100% Category 1 Category I 3

Sodium salicylate 54-21-7 Solid 4.84E-12 100% Category 1 Category I 1 Sorbitan monolaurate 1338-39-2 Liquid 1.25E-15 100% Not Classified Category IV 2 Stearyltrimethylammonium chloride 112-03-8 Solid NA 10% Category 1 Category I 3

Styrene 100-42-5 Liquid 0.673 100% Not Classified Category III 1 Sucrose fatty acid ester NA Solid NA 100% ≥Category 2A ≥Category II 2 Toluene 108-88-3 Liquid 3.160 100% ≥Category 2B Category III 1 Triethanolamine 102-71-6 Liquid 4.51E-07 100% Not Classified Category III 1 Triethanolamine polyoxyethylene(3.0) lauryl ether sulfate


Triton X-100 9002-93-1 Liquid 0 100% Category 1 Category I 3 Tween 20 9005-64-5 Liquid 0 100% Not Classified Category III 2 Tween 80 9005-65-6 Liquid 0 100% Not Classified Category IV 1 Xylene 1330-20-7 Liquid 0.883 100% Not Classified Category II 1 Abbreviations: CASRN = CAS Registry Number® (American Chemical Society); EPA = U.S. Environmental Protection Agency; GHS = United Nations Globally Harmonized

System of Classification and Labelling of Chemicals; JaCVAM = Japanese Center for the Validation of Alternative Methods; kPa = kilopascals; NA = not available; STE = short time exposure.


22

a A bottom-up approach is used to distinguish GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours) and STE Rank 1 from all other hazard categories (i.e., GHS Category 1, 2A, 2B; EPA Category I, II, III; or STE Rank 2 and 3).

b Vapor pressure is expressed in kilopascals at 25°C. Vapor pressures were found using the Hazardous Substances Data Bank (HSDB® [U.S. National Library of Medicine]), available at http://toxnet.nlm.nih.gov (accessed 2/25/2013) or from ChemSpider (available at www.chemspider.com [accessed 2/25/2013]). If actual values were not available, predicted values were obtained from the U.S. EPA EPI (Estimation Programs Interface) Suite™ for Microsoft® Windows, v. 4.11) or ACD/Labs’ ACD/PhysChem Suite available at http://www.acdlabs.com/products/pc_admet/physchem/physchemsuite/ (accessed 2/25/2013). Data from the EPI Suite and ACD/PhysChem Suite programs were also available in ChemSpider.

c The concentration as tested in the rabbit eye test, based on NICEATM data. For substances tested at 100%, the starting material was tested neat/undiluted. d The consensus classification of two or more studies. When there was no consensus using either the GHS (UN 2011) or EPA (EPA 2012) eye hazard classification system (e.g.,

one GHS Category 2A and one GHS Category 2B), the more hazardous classification (i.e., GHS Category 2A) was used as the consensus classification. e STE rank scores from Kao Corporation were equated to the GHS or EPA classification of eye hazard (i.e., UN 2011 and EPA 2012) such that an STE rank of 3 was considered a

severe eye irritant or corrosive (i.e., GHS Category 1 or EPA Category I); an STE rank of 2 was considered a moderate to mild eye irritant (i.e., GHS Category 2A or 2B or EPA Category II or III); and an STE rank of 1 was considered to be equivalent to GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours).

Table 2-3 Test Substances Used to Evaluate STE Performance in a Bottom-Up Approach Within the Defined Applicability Domaina,b

Substance CASRN App Domainb

Physical Form as Tested

Vapor Pressure

(kPa 25ºC)c

NICEATM In Vivo Concd

(%)

GHS-NICEATM Consensuse

EPA-NICEATM Consensuse

JaCVAM Overall

Consensus STE Rankf

1-Bromo-4-chlorobutane 6940-78-9 1,2 Liquid 1.640E-01 100% Not classified Category IV 1

1-Dodecanaminium, N-(2-hydroxy-3-sulfopropyl)-N,N-dimethyl-, inner salt

13197-76-7 1,2 Liquid 1.160E-21 neat Category 1 Category I 2

1-Methylpropyl benzene 135-98-8 1,2 Liquid 1.760E-01 100% Not classified Category IV 1

1-Octanol 111-87-5 1,2 Liquid 1.320E-02 100% Category 2A Category II 2

1,3-Di-isopropylbenzene 99-62-7 1,2 Liquid 4.100E-02 100% Not classified Category IV 1

1,9-Decadiene 1647-16-1 1,2 Liquid 3.200E-01 100% Not classified Category IV 1

2-Benzyloxyethanol 622-08-2 1,2 Liquid 2.940E-04 100% Category 2A Category II 2

2-Ethoxyethyl acetate (Cellosolve acetate) 111-15-9 1,2 Liquid 3.970E-01 100% Not classified Category III 1

2-Ethyl-1-hexanol 104-76-7 1,2 Liquid 2.460E-02 100% Category 2A Category II 2



23



Vapor Pressure

(kPa 25ºC)c


(%)



JaCVAM Overall

Consensus STE Rankf

2-Ethylhexyl p-dimethylamino benzoate 21245-02-3 1,2 Liquid 4.720E-06 100% Not classified Category IV 1

2-Methyl-1-pentanol 105-30-6 1,2 Liquid 1.910E-01 100% Category 2B Category III 2

2-Methylbutyric acid 116-53-0 1,2 Liquid 1.490E-01 100% Category 1 Category I 2

2-Naphthalenesulfonic acid,6-hydroxy-,monosodium salt, polymer with formaldehyde and hydroxymethylbenzenesulfonic aid monosodium salt

85255-76-1 1,2 Liquid NA neat Category 1 Category II 2

2,2-Dimethyl-3-pentanol 3970-62-5 1,2 Liquid 4.130E-01 100% Not classified Category III 1

2,4-Pentanediol 625-69-4 1,2 Liquid 7.300E-03 100% Not classified Category IV 1

3-Methoxy-1,2-propanediol 623-39-2 1,2 Liquid 1.920E-03 100% Not classified Category IV 1

Acid red 92 18472-87-2 1 Solid 5.710E-24 100% Category 1 Category I 3

Acrylic acid homopolymer sodium salt 9003-04-7 2 Solid 4.560E-04 neat Not classified Category IV 1

Benzalkonium chloride 8001-54-5 1,2 Liquid NA 100% Category 1 Category I 3

Benzalkonium chloride (10%) 63449-41-2 2 Solid NA 10 Category 1 Category I 3

Benzene, 1,1'-oxybis-, tetrapropylene derivs., sulfonated, sodium salts

119345-04-9 2 Solid NA neat Category 1 Category I 3

Benzyl alcohol 100-51-6 1,2 Liquid 7.140E-03 100% Category 1 Category I 2

Body shampoo A NA 1,2 Liquid NA 100 Category 2A Category II 2

Butanol 71-36-3 1,2 Liquid 1.040E+00 100% Category 1 Category I 2

Butyl acetate 123-86-4 1,2 Liquid 1.587E+00 100% Not classified Category III 1


24



Vapor Pressure

(kPa 25ºC)c


(%)



JaCVAM Overall

Consensus STE Rankf

Butyl cellosolve 111-76-2 1,2 Liquid 6.330E-02 100% Category 1 Category II 2

Butylnaphthalenesulfonic acid sodium salt 25638-17-9 2 Solid NA neat Category 1 Category I 2

Butyrolactone 96-48-0 1,2 Liquid 3.940E-02 100% Category 2A Category II 2

Cetylpyridinium bromide (10%) 140-72-7 2 Solid 3.470E-07 10% Category 1 Category I 3

Cetylpyridinium chloride 6004-24-6 2 Solid NA 10% Category 1 Category I 3

Cetyltrimethylammonium bromide (10%) 57-09-0 2 Solid NA 10% Category 1 Category I 3

Calcium thioglycolate 5793-98-6 1 Solid 4.200E-03 100% Category 1 Category I 2

Cyclohexanol 108-93-0 1,2 Liquid 8.660E-02 100% Category 1 Category I 2

Cyclohexanone 108-94-1 1,2 Liquid 5.390E-01 100% Not classified Category III 2

Cyclopentanol 96-41-3 1,2 Liquid 3.070E-01 100% Category 2B Category II 2

Di-n-propyl disulphide 629-19-6 1,2 Liquid 6.640E-02 100% Not classified Category IV 1

Di(2-Ethylhexyl) sodium sulfosuccinate 577-11-7 1,2 Solid 1.630E-15 10% Category 1 Category I 3

Di(propylene glycol) propyl ether 29911-27-1 1,2 Liquid 2.380E-04 100% Category 2B Category III 2

Diethylethanolamine 100-37-8 1,2 Liquid 8.630E-02 100% Category 1 Category I 2

Diisobutyl ketone 108-83-8 1,2 Liquid 2.870E-01 100% Not classified Category IV 1

Dimethyl sulfoxide 67-68-5 1,2 Liquid 8.290E-02 100% Not classified Category III 1

Distearyldimethylammonium chloride 107-64-2 1,2 Solid 2.550E-15 100% Category 1 Category I 2

Dodecane 112-40-3 1,2 Liquid 3.150E-02 100% Not classified Category III 1


25



Vapor Pressure

(kPa 25ºC)c


(%)



JaCVAM Overall

Consensus STE Rankf

Domiphen bromide 538-71-6 1,2 Solid NA 10% Category 1 Category I 3

Ethyl 2-methylacetoacetate 609-14-3 1,2 Liquid 9.150E-02 100% Category 2B Category III 2

Ethyl trimethyl acetate 3938-95-2 1,2 Liquid 2.240E+00 100% Not classified Category III 1

Ethylhexyl salicylate 118-60-5 1,2 Liquid 9.510E-07 100% Not classified Category IV 1

Glycerol 56-81-5 1,2 Liquid 1.060E-05 100% Not classified Category IV 1

Glycidyl methacrylate 106-91-2 1,2 Liquid 8.290E-02 100% Not classified Category III 2

Imidazole 288-32-4 1 Solid 5.780E-04 100% Category 1 Category I 2

Iso-octyl acrylate 29590-42-9 1,2 Liquid 2.040E-02 100% Not classified Category IV 1

Isobutyl alcohol 78-83-1 1,2 Liquid 1.780E+00 100% Category 1 Category I 2

Isopropyl myristate 110-27-0 1,2 Liquid 1.080E-04 100% Not classified Category IV 1

Lactic acid 50-21-5 1,2 Liquid 3.810E-03 100% Category 1 Category I 2

Lauric acid 143-07-7 1,2 Solid 2.130E-09 neat ≥Category 2A ≥Category II 2

Lauryldimethylamine oxide 1643-20-5 2 Solid 1.680E-15 neat Category 1 Category I 3

Lotion A NA 1,2 Liquid NA 100 Not classified Category IV 1

m-Phenylene diamine 108-45-2 1 Solid 2.510E-04 100% Category 1 Category I 2

Methoxyethyl acrylate 3121-61-7 1,2 Liquid 5.980E-01 100% Category 1 ≥Category III 2

Methyl amyl ketone 110-43-0 1,2 Liquid 6.550E-01 100% Not classified Category III 1

Methyl cyanoacetate 105-34-0 1,2 Liquid 4.690E-02 100% Category 2A Category II 2

Methyl isobutyl ketone 108-10-1 1,2 Liquid 2.900E+00 100% Not classified Category III 1

Methyl trimethyl acetate 598-98-1 1,2 Liquid 4.760E+00 100% Not classified Category IV 1


26



Vapor Pressure

(kPa 25ºC)c


(%)



JaCVAM Overall

Consensus STE Rankf

n-Hexanol 111-27-3 1,2 Liquid 1.170E-01 100% Category 2A Category II 2

n-Hexyl bromide 111-25-1 1,2 Liquid 5.410E-01 100% Not classified Category IV 1

n-Octyl bromide 111-83-1 1,2 Liquid 6.910E-02 100% Not classified Category IV 1


188070-49-7 2 Solid NA neat Category 2A Category II 2

n,n-Dimethylguanidine sulfate 598-65-2 1 Solid 4.040E+00 100% Not classified Category III 1

Polyethylene glycol 400 25322-68-3 1,2 Liquid NA 100% Not classified Category IV 1

Polyethyleneglycol monolaurate (10 E.O.) 9004-81-3 1,2 Liquid 0.000E+00 100% Not classified Category IV 2

Polyoxyethylene hydrogenated castor Oil (60E.O.) 61788-85-0 2 Solid NA 100% Not classified Category IV 1

Polyoxyethylene(10) polyoxypropylene(1.5) lauryl-myristyl ether 68439-51-0 1,2 Liquid NA neat Category 1 Category I 3

Polyoxyethylene(13) (mono-, di-, tri-)styrenated phenyl ether 104376-75-2 1,2 Liquid NA neat Not classified Category III 3

Polyoxyethylene(14) tribenzylated phenyl ether 116998-28-8 1,2 Liquid NA neat Not classified Category IV 1

Polyoxyethylene(160) sorbitan triisostearate 54392-28-8 1,2 Solid NA neat Not classified Category IV 1

Polyoxyethylene(19) (mono-, di-, tri-)styrenated phenyl ether 104376-75-2 1,2 Liquid NA neat Not classified Category II 2

Polyoxyethylene(20) hydrogenated tallow amine 61790-82-7 1,2 Solid NA neat ≥Category

2A ≥Category II 3

Polyoxyethylene(23) lauryl ether 9002-92-0 2 Solid 2.030E-13 neat Category 2A Category III 2


27



Vapor Pressure

(kPa 25ºC)c


(%)



JaCVAM Overall

Consensus STE Rankf

Polyoxyethylene(40) hydrogenated castor oil 61788-85-0 1,2 Liquid NA neat Not classified Category IV 1

Potassium laurate 10124-65-9 1,2 Solid 0.000E+00 10% Category 1 Category I 3

Potassium oleate 143-18-0 2 Solid 4.930E-10 neat Not classified Category III 2

Promethazine hydrochloride 58-33-3 1 Solid 0.000E+00 100% Category 1 Category I 3

Propasol solvent P 1569-01-3 1,2 Liquid 1.800E-01 100% Category 2B Category II 2

Propylene glycol 57-55-6 1,2 Liquid 1.480E-02 100% Not classified Category IV 1

Pyridine 110-86-1 1,2 Liquid 2.580E+00 100% Category 1 Category I 2

Rinse A NA 1,2 Liquid NA 100 Not classified Category III 2

Rinse B NA 1,2 Liquid NA 100 Category 2B Category III 2

Rinse C NA 1,2 Liquid NA 100 Not classified Category IV 1

Rinse D NA 1,2 Liquid NA 100 Not classified Category III 1

Shampoo A NA 1,2 Liquid NA 100 Category 2A Category II 2

Shampoo B NA 1,2 Liquid NA 100 Category 1 Category I 2

Shampoo C NA 1,2 Liquid NA 100 Category 2A Category II 2

Shampoo D NA 1,2 Liquid NA 100 Category 2A Category II 2

Sodium hydroxide 1310-73-2 1 Solid 6.530E-22 10% Category 1 Category I 3

Sodium lauryl sulfate 151-21-3 2 Solid 2.400E-13 100% ≥Category 2A Category III 3

Sodium lauryl sulfate (15%) 151-21-3 2 Solid NA 15 Category 1 Category I 3

Sodium polyoxyethylene(3) lauryl ether sulfate 9004-82-4 1,2 Liquid 2.270E-13 neat Category 1 Category I 3


28



Vapor Pressure

(kPa 25ºC)c


(%)



JaCVAM Overall

Consensus STE Rankf

Sorbitan monolaurate 1338-39-2 1,2 Liquid 1.250E-15 neat Not classified Category IV 2

Stearyltrimethylammonium chloride 112-03-8 1,2 Solid NA 10% Category 1 Category I 3

Styrene 100-42-5 1,2 Liquid 6.730E-01 100% Not classified Category III 1

Sucrose fatty acid ester NA 1,2 Solid NA 100% ≥Category 2A ≥Category II 2

Toluene 108-88-3 1,2 Liquid 3.160E+00 100% ≥Category 2B Category III 1

Triethanolamine 102-71-6 1,2 Liquid 4.510E-07 100% Not classified Category III 1

Triethanolamine polyoxyethylene(3.0) lauryl ether sulfate

27028-82-6 1,2 Liquid 2.500E-10 neat Category 1 Category I 3

Triton X-100 9002-93-1 1,2 Liquid 0.000E+00 100% Category 1 Category I 3

Tween 20 9005-64-5 1,2 Liquid 0.000E+00 100% Not classified Category III 2

Tween 80 9005-65-6 1,2 Liquid 0.000E+00 100% Not classified Category IV 1

Xylene 1330-20-7 1,2 Liquid 8.826E-01 100% Not classified Category II 1 Abbreviations: App = applicability; CASRN = CAS Registry Number® (American Chemical Society); EPA = U.S. Environmental Protection Agency; GHS = United Nations

Globally Harmonized System of Classification and Labelling of Chemicals; JaCVAM = Japanese Center for the Validation of Alternative Methods; kPa = kilopascals; NA = not available; NICEATM = National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods; STE = short time exposure.

a A bottom-up approach is used to distinguish GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours) and STE Rank 1 from all other hazard categories (i.e., GHS Category 1, 2A, 2B; EPA Category I, II, III; or STE Rank 2 and 3).

b There are two defined applicability domains (AD) for the STE test method. AD 1 (n=94 substances) includes all substances with vapor pressures ≤6 kilopascals but excludes solids that are alcohols, hydrocarbons, or salts. AD 2 (n = 101 substances) includes all liquids with vapor pressures ≤6 kilopascals and solid surfactants or surfactant-containing formulations (i.e., nonsurfactant solids and substances with vapor pressures >6 kilopascals are excluded).

c Vapor pressure is expressed in kilopascals at 25°C. Vapor pressures were found using the Hazardous Substances Data Bank (HSDB® [U.S. National Library of Medicine]), available at http://toxnet.nlm.nih.gov (accessed 2/25/2013) or from ChemSpider (available at www.chemspider.com [accessed 2/25/2013]). If actual values were not available, predicted values were obtained from the U.S. EPA EPI (Estimation Programs Interface) Suite™ for Microsoft® Windows, v. 4.11) or ACD/Labs’ ACD/PhysChem Suite available at http://www.acdlabs.com/products/pc_admet/physchem/physchemsuite/ (accessed 2/25/2013). Data from the EPI Suite and ACD/PhysChem Suite programs were also available in ChemSpider.



29

d The concentration as tested in the rabbit eye test, based on NICEATM data. e The consensus classification of two or more studies. When there was no consensus using either the GHS (UN 2011) or EPA (EPA 2012) eye hazard classification system (e.g.,

one GHS Category 2A and one GHS Category 2B), the more hazardous classification (i.e., GHS Category 2A) was used as the consensus classification. f STE rank scores were equated to the GHS or EPA classification of eye hazard (i.e., UN 2011 and EPA 2012) such that an STE rank of 3 was considered a severe eye irritant or

corrosive (i.e., GHS Category 1 or EPA Category I); an STE rank of 2 was considered a moderate to mild eye irritant (i.e., GHS Category 2A or 2B or EPA Category II or III); and an STE rank of 1 was considered to be equivalent to GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours).


30

3.0 STE Test Method Performance Test method performance is typically evaluated by calculating the following (ICCVAM 2003):

• Accuracy (concordance): the proportion of correct outcomes (positive and negative) of a test method

• Sensitivity: the proportion of all positive substances that are classified correctly as positive • Specificity: the proportion of all negative substances that are classified correctly as negative • Positive predictivity: the proportion of correct positive responses among substances testing

positive • Negative predictivity: the proportion of correct negative responses among substances testing

negative • False positive rate: the proportion of all negative substances that are falsely identified as positive • False negative rate: the proportion of all positive substances that are falsely identified as negative

The STE test method performance was evaluated for each study, and the data set is provided in Supplement B. An overall STE ocular irritation classification was assigned for each test substance in the database based on the majority of ocular irritation classification calls. When a test substance had an even number of different irritation classifications (e.g., two tests classified a substance as a moderate irritant and two tests classified a substance as a severe irritant), the more severe hazard classification was used for its overall classification (e.g., severe irritant). Using the consensus ocular irritation classification for each substance, the STE test method was evaluated in a top-down approach to distinguish ocular corrosives and severe irritants (i.e., GHS Category 1 or EPA Category I) from all other categories (i.e., GHS Category 2A, 2B, Not Classified or EPA Category II, III, IV). The STE test method was also evaluated in a bottom-up approach to identify GHS Not Classified substances or EPA Category IV (minimally irritant) substances from all other irritant categories (i.e., GHS Category 1, 2A, or 2B or EPA Category I, II, or III).

The overall accuracy of the STE test method in a top-down approach ranged from 70% to 96%, and the accuracy in a bottom-up approach ranged from 80% to 85% depending on the classification. The predictive capacity of the STE test method was assessed by identifying the chemical classes or physical properties that increased the false positive rate in a top-down approach and those that increased the false negative rate in a bottom-up approach. Excluding discordant chemical classes or physical properties optimized the applicability domain for a top-down or bottom-up approach.

3.1 GHS Classification System: STE Performance in a Top-Down Approach

The performance of the STE test method was evaluated for GHS ocular hazard classification in a top-down approach. STE accuracy, sensitivity, specificity, false positive rate, and false negative rate were determined based on available in vivo reference data for the test substances. Test substances that were identified as direct MTT reducers were removed from the analyses. These include two substances from Kojima et al. (Kao BRD), two substances from Takahashi et al. (2010), and one substance from the Kao in-house studies. These analyses were performed for each of the five studies as well as for 120 unique substances from these five studies that remained after duplicates were removed and consensus classifications were assigned (Table 3-1). The GHS classification for each test substance is listed in Supplement B.


31

Table 3-1 STE Performance for GHS Classification in a Top-Down Approach

Data Source N Accuracy Sensitivity Specificity False

Positive Ratea

False Negative Rateb

% No.c % No.c % No.c % No.c % No.c Kojima et al. (Kao BRD) 30 70 21/30 10 1/10 100 20/20 0 0/20 90 9/10

Sakaguchi et al. 2011 23 96 22/23 80 4/5 100 18/18 0 0/18 20 1/5

Takahashi et al. 2009 37 84 31/37 65 11/17 100 20/20 0 0/20 35 6/17

Takahashi et al. 2010 47 83 39/47 58 11/19 100 28/28 0 0/28 42 8/19

Kao In-House 22 96 21/22 0 0/1 100 21/21 0 0/21 100 1/1

Kao New Surfactants 39 69 27/39 45 9/20 95 18/19 5.3 1/19 55 11/20

Unique Substancesd 120 85 102/120 53 19/36 99 83/84 1.2 1/84 47 17/36

Abbreviations: BRD = background review document; GHS = Globally Harmonized System of Classification and Labelling of Chemicals (UN 2011); N = number of substances; STE = short time exposure.

a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage. d Substances from all of the above studies remaining after duplicates were removed and consensus classifications were

assigned.

3.1.1 STE Discordant Results for GHS Classification in a Top-Down Approach

The STE results that were discordant with in vivo results were analyzed further. These analyses were performed on specific categories of chemicals, as well as on certain physicochemical properties potentially relevant to ocular toxicity testing (e.g., surfactants, pH, physical form).

Several trends were noted in STE performance among these subgroups of substances (Table 3-2). Only one of 84 substances was overpredicted (i.e., false positive) and slightly affected the overprediction of its constituent chemical classes (3.6% to 8.3% overprediction). The chemical categories of substances that were most consistently underpredicted (i.e., false negatives) by the STE test method were alcohols and carboxylic acids. Of the 17 underpredicted substances, 7 were alcohols, 4 were carboxylic acids, and 3 were salts. Additional chemical categories represented among the underpredicted substances were esters (2) and heterocyclic compounds (2).

With regard to the physical form of the substances underpredicted by the STE test method, 12 were liquids and 5 were solids. Considering the proportion of the total available database, solids (16%; 5/31) and liquids (13%; 12/89) were underpredicted at a similar rate by the STE test method.


32

Table 3-2 STE False Positive and False Negative Rates by Chemical Category and Properties of Interest for GHS Classification in a Top-Down Approach

Category N False Positive Ratea False Negative Rateb % No.c % No.c

Overall 120 1.2 1/84 47 17/36 Chemical Categoryd Alcohol 39 3.6 1/28 64 7/11 Amine/Amidine 8 0 0/2 17 1/6 Carboxylic acid 21 0 0/14 57 4/7 Ester 17 0 0/14 67 2/3 Ether/Polyether 16 8.3 1/12 0 0/4 Heterocyclic compound 9 0 0/3 33 2/6 Hydrocarbon 23 5.0 1/20 33 1/3 Ketone 8 0 0/8 - 0/0 Onium compound 10 0 0/1 11 1/9 Salt 17 0 0/6 27 3/11 Properties of Interest Liquids 89 1.4 1/72 71 12/17 Solids 31 0 0/12 26 5/19 Surfactants – Total -nonionic -anionic -cationic -ampholyic

44 14 11 7 2

4.2 8.3 0 - -

1/24 1/12 0/3 0/0 0/0

20 0 25 0 50

4/20 0/2 2/8 0/7 1/2

pH – Totale -acidic (pH < 7.0) -basic (pH > 7.0) -equals 7

27 19 7 1

0 0 0 0

0/10 0/8 0/1 0/1

41 36 50 -

7/17 4/11 3/6 0/0

Vapor Pressure – Total >6kPa ≤6kPa

90 13 77

0 0 0

0/66 0/13 0/53

58 -

58

14/24

0/0 14/24

Abbreviations: GHS = Globally Harmonized System of Classification and Labelling of Chemicals (UN 2011); kPa = kilopascals; N = number of substances; STE = short time exposure.

a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage. d One or more chemical categories were assigned to each test substance based on the chemical categories outlined in the

tree structure provided for that chemical in the National Library of Medicine’s Medical Subject Headings (MeSH®) for inorganic or organic chemicals when available (http://www.nlm.nih.gov/mesh) and on the presence of common organic functional groups (i.e., ketones) if that functional group was not available in the MeSH tree structure.

e Total number of substances with pH data available.

http://www.nlm.nih.gov/mesh


33

Table 3-3 shows the STE test method performance in a top-down approach when problematic categories are excluded that gave the most discordant results in the GHS classification system. In general, exclusion of alcohols, ethers/polyethers, hydrocarbons, or nonionic surfactants individually reduced false positive rates to 0% and marginally reduced or slightly increased false negative rates. The performance of validated in vitro methods for GHS classification is included for comparison.

Table 3-3 STE Performance for GHS Classification in a Top-Down Approach After Excluding Discordant Categories

Method Evaluated Accuracy False Positive Ratea False Negative Rateb

% No.c % No.c % No.c STE Overall 85 102/120 1.2 1/84 47 17/36 STE w/o Alcohols 87 71/82 0 0/56 42 11/26 STE w/o Ethers/Polyethers 84 87/104 0 0/72 53 17/32 STE w/o Hydrocarbons 84 81/97 0 0/64 49 16/33 STE w/o Nonionic surfactants 84 89/106 0 0/73 50 17/34

BCOP 79 149/188 24 29/123 15 10/65 ICE 83 120/144 8 9/114 50 15/30 CM 90 74/82 2 1/48 21 7/34 Abbreviations: BCOP = bovine corneal opacity and permeability; CM = Cytosensor microphysiometer; GHS = Globally

Harmonized System of Classification and Labelling of Chemicals (UN 2011); ICE = isolated chicken eye; N = number of substances; STE = short time exposure.

a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage.

3.2 GHS Classification System: STE Performance in a Bottom-Up Approach

The performance of the STE test method was evaluated for GHS ocular hazard classification in a bottom-up approach. STE accuracy, sensitivity, specificity, false positive rate, and false negative rate were determined based on available in vivo reference data for the test substances. Test substances that were identified as direct MTT reducers and classified as STE nonirritants were removed from the bottom-up analysis, as these could be false negative. These include two substances from Kojima et al. (Kao BRD), two substances from Takahashi et al. (2010), and one substance from the Kao in-house studies. These analyses were performed for each of the five studies as well as for 129 unique substances from these five studies that remained after duplicates were removed and consensus classifications were assigned (Table 3-4). The GHS classification for each test substance is listed in Supplement B.


34

Table 3-4 STE Performance for GHS Classification in a Bottom-Up Approach


Positive Ratea

False Negative

Rateb


Sakaguchi et al. 2011 24 88 21/24 77 10/13 100 11/11 0 0/11 23 3/13

Takahashi et al. 2009 39 87 34/39 85 23/27 92 11/12 8.3 1/12 15 4/27

Takahashi et al. 2010 52 83 43/52 85 28/33 79 15/19 21 4/19 15 5/33

Kao In-House 22 96 21/22 100 1/1 95 20/21 4.8 1/21 0 0/1

Kao New Surfactants 34 85 29/34 100 22/22 58 7/12 42 5/12 0 0/22

Unique Substancesd 129 85 109/129 88 64/73 80 45/56 20 11/56 12 9/73

Abbreviations: BRD = background review document; GHS = Globally Harmonized System of Classification and Labelling of Chemicals (UN 2011); N = number of substances; STE = short time exposure.

a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage. d Substances from all of the above studies remaining after duplicates were removed and consensus classifications were

assigned.

3.2.1 STE Discordant Results for GHS Classification in a Bottom-Up Approach


Several trends were noted in STE performance among these subgroups of substances (Table 3-5). The overall false positive rate was 20%. The chemical categories of substances that the STE test method most consistently underpredicted for GHS classification (i.e., false negatives) were salts (13%; 2/15), hydrocarbons (33%; 2/6), and alcohols (16%; 4/25).

With regard to the physical form of the substances underpredicted by the STE test method, four were liquids and five were solids. Considering the proportion of the total available database, solids (14%; 5/37) appear more likely than liquids (4.3%; 4/92) to be underpredicted by the STE test method.


35

Table 3-5 STE False Positive and False Negative Rates by Chemical Category and Properties of Interest for GHS Classification in a Bottom-Up Approach


Overall 129 20 11/56 12 9/73 Chemical Categoryd Alcohol 41 31 5/16 16 4/25 Amine/Amidine 17 0 0/2 0 0/15 Carboxylic acid 33 28 3/11 9.1 2/22 Ester 18 46 5/11 14 1/7 Ether/Polyether 16 38 3/8 0 0/8 Heterocyclic compound 10 50 1/2 0 0/8 Hydrocarbon 24 17 3/18 33 2/6 Ketone 8 20 1/5 0 0/3 Onium compound 12 - 0/0 8.3 1/12 Salt 18 67 2/3 13 2/15 Properties of Interest Liquids 92 18 9/50 9.5 4/42 Solids 37 33 2/6 16 5/31 Surfactants – Total -nonionic -anionic -cationic

49 16 12 8

41 46 50 -

7/17 5/11 1/2 0/0

0 0 0 0

0/32 0/5

0/10 0/8


33 23 8 1

25 17 0 0

2/8 1/6 0/1 0/1

13 18 0 -

3/24 3/17 0/7 0/0


97 14 83

16 14 16

7/44 1/7

6/37

17 43 13

9/53 3/7

6/46 Abbreviations: GHS = Globally Harmonized System of Classification and Labelling of Chemicals (UN 2011);

kPa = kilopascals; N = number of substances; STE = short time exposure. a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = The proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage. d One or more chemical categories were assigned to each test substance based on the chemical categories outlined in the


e Total number of substances with pH data available. Table 3-6 shows the STE test method performance in a bottom-up approach when problematic categories are excluded that gave the most discordant results in the GHS classification system. In



36

general, exclusion of alcohols, hydrocarbons, salts, or solids individually resulted in small changes in assay performance. However, two applicability domains were evaluated based on excluding certain chemical and product classes, or physical characteristics. When substances with high vapor pressures, solid alcohols, hydrocarbons, and salts were excluded, the false negative rate was reduced to 2.0% (1/49). When substances with high vapor pressures and nonsurfactant solids were excluded, the false negative rate was reduced to 1.9% (1/54). The single false negative substance using the restricted applicability domains was toluene. In the NICEATM database, an in vivo study from the European Centre for Ecotoxicology and Toxicology of Chemicals classifies toluene as GHS Not Classified (ECETOC 1998), whereas a study submitted to the EPA under the Toxic Substances Control Act (TSCA) classifies it as GHS Category 2B (eye irritation data made available by the U.S. Environmental Protection Agency). These data suggest toluene is a mild ocular irritant and mitigates concern about the false negative classification. The performance of validated in vitro methods for GHS classification is included in Table 3-6 for comparison.

Table 3-6 STE Performance for GHS Classification in a Bottom-Up Approach After Excluding Discordant Categories


% No.c % No.c % No.c STE Overall 85 109/129 20 11/56 12 9/73 STE w/o Alcohols 89 78/89 15 6/40 10 5/49 STE w/o Hydrocarbons 86 90/105 21 8/38 10 7/67 STE w/o Salts 86 95/111 17 9/53 12 7/58 STE w/o Solids 86 79/92 18 9/50 9.5 4/42 STE w/o Vapor Pressure >6kPa, solid alcohols, hydrocarbons, and salts

90 85/94 18 8/45 2.0 1/49

STE w/o Vapor Pressure >6kPa and Nonsurfactant Solids 90 91/101 19 9/47 1.9 1/54

BCOP 66 125/188 69 63/91 0 0/97 CM 68 36/53 68 17/25 0 0/28 Abbreviations: BCOP = bovine corneal opacity and permeability; CM = Cytosensor microphysiometer; GHS = Globally

Harmonized System of Classification and Labelling of Chemicals (UN 2011); kPa = kilopascals; N = number of substances; STE = short time exposure.


3.3 EPA Classification System: STE Performance in a Top-Down Approach

The performance of the STE test method was evaluated for EPA ocular hazard classification in a top-down approach. STE accuracy, sensitivity, specificity, false positive rate, and false negative rate were determined based on available in vivo reference data for the test substances. Test substances that were identified as direct MTT reducers were removed from the analyses. These include two substances from Kojima et al. (Kao BRD), two substances from Takahashi et al. (2010), one substance from the Kao in-house studies, and five substances from the combined overall data set. These analyses were performed for each of the five studies as well as for the overall data set of 120 test substances from all these studies (Table 3-7). The EPA classification for each test substance is listed in Supplement B.


37

Table 3-7 STE Performance for EPA Classification in a Top-Down Approach

Data Source N Accuracy Sensitivity Specificity False Positive

Ratea False Negative

Rateb

% No.c % No.c % No.c % No.c % No.c Kojima et al. (Kao BRD)

30 70 21/30 11 1/9 95 20/21 4.8 1/21 89 8/9

Sakaguchi et al. 2011 24 92 22/24 67 4/6 100 18/18 0 0/18 33 2/6

Takahashi et al. 2009 38 82 31/38 61 11/18 100 20/20 0 0/20 39 7/18

Takahashi et al. 2010 49 82 40/49 58 11/19 97 29/30 3.3 1/30 42 8/19

Kao In-House 21 100 21/21 - 0/0 100 21/21 0 0/21 - 0/0

Kao New Surfactants 39 72 28/39 47 9/19 95 19/20 5.0 1/20 53 10/19

Overall 120 87 104/120 58 19/33 98 85/87 2 2/87 42 14/33

Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); N = number of substances; STE = short time exposure.


3.3.1 STE Discordant Results for EPA Classification in a Top-Down Approach


Several trends were noted in STE performance among these subgroups of substances (Table 3-8). Two of 87 substances were overpredicted (i.e., false positives) and affected its representative chemical classes, with false positive rates ranging from 4.8% to 12.5%. The chemical categories of substances that were most consistently underpredicted for EPA classification (i.e., false negatives) were alcohols (64%; 7/11) and carboxylic acids (50%; 3/6). Of the 14 underpredicted substances, seven were alcohols, three were carboxylic acids, two were heterocyclic compounds, and two were salts.

With regard to the physical form of the substances overpredicted by the STE test method, 1.4% (1/74) were liquids and 7.7% (1/13) were solids. With regard to the physical form of the substances underpredicted by the STE test method, 10 were liquids and 5 were solids. Considering the proportion of the total available database, solids (16%; 5/32) appear more likely than liquids (11%; 10/89) to be underpredicted by the STE test method.


38

Table 3-8 STE False Positive and False Negative Rates by Chemical Category and Properties of Interest for EPA Classification in a Top-Down Approach


Overall 120 2.3 2/87 42 14/33 Chemical Categoryd Alcohol 40 6.9 2/29 64 7/11 Amine/Amidine 8 0 0/2 17 1/6 Carboxylic acid 20 0 0/14 50 3/6 Ester 16 0 0/15 0 0/1 Ether/Polyether 16 8.3 1/12 0 0/4 Heterocyclic compound 9 0 0/3 33 2/6 Hydrocarbon 24 4.8 1/21 33 1/3 Ketone 8 0 0/8 - 0/0 Onium compound 10 0 0/1 11 1/9 Salt 18 12.5 1/8 20 2/10 Properties of Interest Liquids 89 1.4 1/74 67 10/15 Solids 32 7.7 1/13 26 5/19 Surfactants – Total -nonionic -anionic -cationic -ampholytic

45 14 12 7 2

7.7 8.3 20 - -

2/26 1/12 1/5 0/0 0/0

16 0 14 0 50

3/19 0/2 1/7 0/7 1/2


28 20 7 1

10 13 0 0

1/10 1/8 0/1 0/1

44 42 50 -

8/18 5/12 3/6 0/0


91 13 78

1.5 0

1.8

1/68 0/12 1/56

57 100 55

13/23 1/1

12/22 Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); kPa = kilopascals; N = number of substances;

STE = short time exposure. a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage. d One or more chemical categories were assigned to each test substance based on the chemical categories outlined in the


e Total number of substances with pH data available. Table 3-9 shows the STE test method in a top-down approach when problematic categories are excluded that gave the most discordant results for the EPA classification system. Exclusion of



39

alcohols reduced the false positive rate from 2.3% (2/87) to 0% (0/58) with only a slight reduction in the false negative rate (42%, 14/33 to 38%, 9/24). In general, removal of other individual chemical classes with false positive rates produced higher false negative rates.

Table 3-9 STE Performance for EPA Classification in a Top-Down Approach After Excluding Discordant Categories


% No.c % No.c % No.c STE Overall 87 104/120 2.3 2/87 42 14/33 STE w/o Alcohols 89 73/82 0 0/58 38 9/24 STE w/o Ethers/Polyethers 85 89/105 1.3 1/75 50 15/30 STE w/o Hydrocarbons 85 82/97 1.5 1/66 45 14/31 STE w/o Salts 86 89/103 1.3 1/79 54 13/24 STE w/o Solids 88 78/89 1.4 1/74 67 10/15 Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); STE = short time exposure. a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage.

3.4 EPA Classification System: STE Performance in a Bottom-Up Approach

The performance of the STE test method was evaluated for EPA ocular hazard classification in a bottom-up approach to identify EPA Category IV substances (i.e., minimal effects clearing in less than 24 hours). STE accuracy, sensitivity, specificity, false positive rate, and false negative rate were determined based on available in vivo reference data for the test substances. Test substances that were identified as direct MTT reducers and classified as STE nonirritants were removed from bottom-up analysis, as these could be false negative. These include two substances from Kojima et al. (Kao BRD) and two substances from Takahashi et al. (2010). These analyses were performed for each of the five studies as well as for the overall data set of 129 test substances from all these studies (Table 3-10). The EPA classification for each test substance is listed in Supplement B.

Table 3-10 STE Performance for EPA Classification in a Bottom-Up Approach


Positive Ratea

False Negative Rateb


Sakaguchi et al. 2011 24 67 16/24 56 10/18 100 6/6 0 0/6 44 8/18

Takahashi et al. 2009 39 80 31/39 75 24/32 100 7/7 0 0/7 25 8/32

Takahashi et al. 2010 52 77 40/52 74 31/42 90 9/10 10 1/10 26 11/42

Kao In-House 22 68 15/22 22 2/9 100 13/13 0 0/13 78 7/9


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Kao New Surfactants 34 94 32/34 96 26/27 86 6/7 14 1/7 3.7 1/27

Overall 129 80 103/129 75 73/97 94 30/32 6.3 2/32 25 24/97

Abbreviations: BRD = background review document; EPA = U.S. Environmental Protection Agency (EPA 2012); N = number of substances; STE = short time exposure.


3.4.1 STE Discordant Results for EPA Classification in a Bottom-Up Approach


Several trends were noted in STE performance among these subgroups of substances (Table 3-11). Two substances were overpredicted. The overall false positive rate was 6.3% (2/32), with alcohols (20% 2/10), esters (33%; 2/6), and heterocyclic compounds (50%; 1/2) overpredicted. The chemical categories of substances that the STE test method most consistently underpredicted for EPA classification (i.e., false negatives) were hydrocarbons, ketones, and esters. Of the 24 underpredicted substances, seven were alcohols, five were carboxylic acids, and six were hydrocarbons. Additional chemical categories represented among the underpredicted substances were amines/amidines (2), esters (3), ethers/polyethers (1), ketones (3), onium compounds (1), and salts (2).

With regard to the physical form of the substances in a bottom-up approach, two liquids were overpredicted (6.9%; 2/29), which was 2.2% (2/92) of the entire database. With regard to the physical form of the substances underpredicted by the STE test method, 18 were liquids and six were solids. Considering the proportion of the total available database, liquids (20%; 18/92) appear more likely than solids (16%; 6/37) to be underpredicted by the STE test method.

Table 3-11 STE False Positive and False Negative Rates by Chemical Category and Properties of Interest for EPA Classification in a Bottom-Up Approach


Overall 129 6.3 2/32 25 24/97 Chemical Categoryd Alcohol 41 20 2/10 23 7/31 Amine/Amidine 11 - 0/0 18 2/11 Carboxylic acid 33 0 0/5 18 5/28 Ester 18 33 2/6 25 3/12 Ether/Polyether 16 0 0/4 8.3 1/12 Heterocyclic compound 10 50 1/2 0 0/8 Hydrocarbon 24 0 0/11 46 6/13 Ketone 8 - 0/0 38 3/8 Onium compound 12 - 0/0 8.3 1/12 Salt 18 0 0/1 12 2/17


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Properties of Interest Liquids 92 6.9 2/29 29 18/63 Solids 37 0 0/3 18 6/34 Surfactants – Total -nonionic -anionic -cationic

49 16 12 8

18 25 0 -

2/11 2/8 0/1 0/0

2.6 0 0 0

1/38 0/8

0/11 0/8


32 23 8 1

17 17 - -

1/6 1/6 0/0 0/0

15 18 12

100

4/26 3/17 1/8 1/1

Vapor Pressure – Total >6kPa <6kPa

97 14 83

8.0 0

9.5

2/25 0/4

2/21

32 50- 29

23/72 5/10

18/62 Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); kPa = kilopascals; N = number of substances; STE = short time exposure. a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage. d One or more chemical categories were assigned to each test substance based on the chemical categories outlined in the


e Total number of substances with pH data available. Table 3-12 shows the STE test method performance in a bottom-up approach when problematic categories are excluded that gave the most discordant results in the EPA classification system. In general, exclusion of alcohols, hydrocarbons, salts, or solids individually resulted in small changes in assay performance. However, two applicability domains were evaluated based on excluding certain chemical and product classes, or physical characteristics. When substances with high vapor pressures, solid alcohols, hydrocarbons, and salts were excluded, the false negative rate was slightly reduced to 21% (14/68). When substances with high vapor pressures, and nonsurfactant solids were excluded, the false negative rate was slightly reduced to 18% (13/73).



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Table 3-12 STE Performance for EPA Classification in a Bottom-Up Approach After Excluding Discordant Categories


% No.c % No.c % No.c STE Overall 80 103/129 6.3 2/32 25 24/97 STE w/o Alcohols 89 72/89 0 0/22 25 17/67 STE w/o Hydrocarbons 81 85/105 9.5 2/21 21 18/84 STE w/o Solids 78 72/92 6.9 2/29 29 18/63 STE w/o Salts 78 87/111 6.5 2/31 28 22/80 STE w/o Vapor Pressure >6kPa, solid alcohols, hydrocarbons, and salts

83 78/94 7.7 2/26 21 14/68

STE w/o Vapor Pressure >6kPa and Nonsurfactant Solids 85 86/101 7.1 2/28 18 13/73

Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); kPa = kilopascals; STE = short time exposure. a False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro. b False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro. c Data used to calculate the percentage.


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4.0 STE Test Method Reliability Test method reliability (intralaboratory repeatability and intra- and interlaboratory reproducibility) is an essential element of an evaluation of assay performance (ICCVAM 2003). Repeatability refers to the closeness of agreement between test results obtained within a single laboratory when the procedure is performed on the same substance under identical conditions within a given time period (ICCVAM 1997, 2003). Intralaboratory reproducibility refers to the extent to which qualified personnel within the same laboratory can replicate results using a specific test protocol at different times. Interlaboratory reproducibility refers to the extent to which different laboratories can replicate results using the same protocol and test chemicals and indicates the extent to which a test method can be transferred successfully among laboratories. A reliability assessment includes (1) reviewing the rationale for selecting the substances used to evaluate test method reliability, (2) discussing the extent to which the substances tested represent the range of possible test outcomes and the properties of the various substances for which the test method is proposed for use, and (3) performing a quantitative and/or qualitative analysis of repeatability and intra- and interlaboratory reproducibility.

Background information, data, and the performance (i.e., accuracy and reliability) analyses of the STE test method conducted by Kao Corporation are provided in the BRD (Supplement A). STE test data were available for replicates within individual experiments repeated three times for each test substance in two to five different laboratories. Coefficient of variation (CV) analyses were performed on within-experiment and between-laboratory STE data, using the cell viability value obtained for each test substance within each of the two to five testing laboratories.

The %CV values for intralaboratory reliability for substances classified as nonirritants ranged from 0.3% to 23.5% in the four studies evaluated. Substances classified in vitro as irritants tended to have greater %CV values, as expected, because the cell viability for these chemicals was often quite low. Further, the mean viability for the positive control, 0.01% sodium lauryl sulfate, was 41.7% (N = 71) with %CV of 24.7%.

In terms of interlaboratory agreement, the laboratories recorded 100% agreement for 83% to 100% of the substances for GHS classification and 87% to 100% of the substances for EPA classification.


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5.0 Peer Review Summary To ensure the completeness of the NICEATM STE performance review, NTP provided the STE Summary Review Document along with the original Kao BRD and other supporting documentation to four external scientific reviewers who were asked to:

• Comment on whether the protocol is complete and adequate

• Comment on the adequacy of the database used for evaluating STE

• Provide any additional published STE studies or data

• Comment on the adequacy of the test method reliability

• Comment on the adequacy of the performance evaluation

• Provide any additional comments on the protocol or analysis

• Provide comments for regulatory agencies considering using data from this test method

The reviewers commented that the evaluation of the STE test method performance was thoroughly conducted. The reviewers remarked that the evaluation not only examined performance of the test method based upon the entire set of chemicals but also had a secondary assessment of STE performance with select chemical classes removed from the applicability domain. Given the thoroughness of the review, the reviewers stated that no other analysis is necessary. As with the performance analysis, the reviewers commented that the reliability analysis was thorough with no need for additional analysis.

The reviewers commented that the STE database was adequate for its intended purpose and added that they were not aware of additional STE data that could be used in this evaluation. The reviewers did however suggest that the STE database would require further development if the test method were to be used in the evaluation of pesticides.

The reviewers made a number of comments directed towards regulatory agencies considering using data obtained from the STE method:

When compared to other in vitro or in vivo assays currently available for eye irritation assessment, STE has a number of advantages, including time and cost required to do the assay, the use of a cell line rather than ex vivo tissue, its ability to assess poorly water-soluble substances, a low false positive rate, and protocol simplicity

The analysis highlights that the performance of a test method is dependent upon the classification system to which it is being compared. Specifically, STE “false negatives”, after all poorly compatible substances are excluded, are EPA Cat III: mild irritants.

Acceptance and use of data from the STE method is suggested by the developers as part of an in vitro / ex vivo battery of tests designed to offer an alternative to the in vivo OECD Test Guideline 405 Acute Eye Irritation/Corrosion assay in rabbits. As such, results from the STE when considered alone would most often be seen as “screening data” and best interpreted as part of a systematic evaluation of hazard.

There are likely to be circumstances in which the predictive nature of the STE assay is unknown (i.e., new chemical domains, mixtures, etc.). Submission of useful STE data relies on careful and


45

consistent conduct of the assay and verification of the performance of the STE method when extended beyond the currently available chemical domain or space.

At present, there is no in vitro alternative test to definitively and accurately distinguish non-irritant and irritant chemicals. In a bottom-up approach aimed at identifying non-irritants, it is important to reduce the false negative substances as much as possible. The false negative rate of BCOP and ICE are 0%, but their false positive rates are high, 68 to 69%. While the false negative rate in STE is not 0%, when either applicability domain is adopted, the rate is 1.9 to 2%. Test systems that obtain high accuracy, low false positive rate, and low false negative rate are desirable and based upon its performance, STE is suitable for use in a bottom-up approach.

As a result of the reviewers comments, minor edits were made to the NICEATM SRD including updating OECD TG references and clarifying the evaluation of direct MTT reducers. The reviewers also had a number of comments and suggestions for the STE protocol that were compiled and provided to Kao Corporation.

STE Peer Reviewers

Michael Joseph Olson

Director, Occupational Toxicology, Hazard Assessment and Communication, Corporate Environment Health and Safety, GlaxoSmithKline

Research Triangle Park, NC

Seung Hyeok Seok

Assistant Professor, Department of Microbiology and Immunology, Institute for Experimental Animals, College of Medicine, Seoul National University

Seoul, Korea

Kristie Sullivan

Director, Regulatory Testing Issues, Physicians Committee for Responsible Medicine

Washington, D.C.

Kohji Yamakage

Chief of Division of alternative toxicology testing, Hatano Research Institute, Food Drug Safety Center

Kanagawa, Japan


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6.0 References Balls M, Berg N, Bruner LH, Curren R, deSilva O, Earl L, et al. 1999. Eye irritation testing: the way forward. Altern Lab Anim 27: 53-77.

Draize J, Woodard G, Calvery H. 1944. Methods for the study of irritation and toxicology of substances applied topically to the skin and mucous membrane. J Pharmacol Exp Ther 83: 377-390.

ECETOC. 1998. Eye Irritation – Reference Chemical Data Bank. 2nd ed. Technical Report No. 48(2). Brussels:European Centre for Ecotoxicology and Toxicology of Chemicals. Available: http://www.ecetoc.org/technical-reports

EPA. 2012. Label Review Manual [Internet]. Available: http://www.epa.gov/oppfead1/labeling/lrm/; last accessed April 8, 2013.

Eskes C, Bessou S, Bruner L, Curren R, Harbell J, Jones P, et al. 2005. 3.3: Eye irritation. Altern Lab Anim 33(Suppl 1): 47-81.

European Union. 2003. Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products. OJ L 66: 26-35. Available: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003L0015:EN:HTML

Huang K, Chen Y, Walker M. 2004. Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids. Biotechniques 37(3): 406-412.

ICCVAM. 1997. Validation and Regulatory Acceptance of Toxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods. NIH Publication No. 97-3981. Research Triangle Park, NC:National Institute of Environmental Health Sciences. Available: http://ntp.niehs.nih.gov/iccvam/docs/about_docs/validate.pdf

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, and Alternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC:National Institute of Environmental Health Sciences. Available: http://ntp.niehs.nih.gov/iccvam/SuppDocs/SubGuidelines/SD_subg034508.pdf

Kojima H, Hayashi K, Sakaguchi H, Omori T, Otoizumi T, Kuwahara H, et al. The second phase validation study of the Short Time Exposure (STE) test to assess the eye irritation potential of chemicals. Manuscript in preparation. (Cited in Kao Corporation BRD Supplement A)

OECD. 2012a. Test No. 437. Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing 4 Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage [revised 21 December, 2012]. In: OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects. Paris: OECD Publishing Available: http://www.oecd.org/env/ehs/testing/BCOP%20TG%20437%202012-12-21_clean.pdf

OECD. 2012b. Test No. 438. Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage [revised 21 December, 2012]. In: OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects. Paris:OECD Publishing Available: http://www.oecd.org/env/ehs/testing/Draft_ICE_TG438_clean_21%20Dec%202012.pdf

Ohno Y, Kaneko T, Inoue T, Morikawa Y, Yoshida T, Fujii A, et al. 1999. Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (1) Overview of the validation study and Draize scores for the evaluation of the tests. Toxicol In Vitro 13(1): 73-98.

http://www.epa.gov/oppfead1/labeling/lrm/

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003L0015:EN:HTML

http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32003L0015:EN:HTML

http://ntp.niehs.nih.gov/iccvam/docs/about_docs/validate.pdf

http://ntp.niehs.nih.gov/iccvam/SuppDocs/SubGuidelines/SD_subg034508.pdf

http://www.oecd.org/env/ehs/testing/BCOP%20TG%20437%202012-12-21_clean.pdf

http://www.oecd.org/env/ehs/testing/Draft_ICE_TG438_clean_21%20Dec%202012.pdf


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Sakaguchi H, Ota N, Omori T, Kuwahara H, Sozu T, Takagi Y, et al. 2011. Validation study of the Short Time Exposure (STE) test to assess the eye irritation potential of chemicals. Toxicol In Vitro 25(4): 796-809.

Sims J, Plattner R. 2009. MTT assays cannot be utilized to study the effects of STI571/Gleevec on the viability of solid tumor cell lines. Cancer Chemother Pharmacol 64: 629-633.

Takahashi Y, Hayashi K, Abo T, Koike M, Sakaguchi H, Nishiyama N. 2011. The Short Time Exposure (STE) test for predicting eye irritation potential: intra-laboratory reproducibility and correspondence to globally harmonized system (GHS) and EU eye irritation classification for 109 chemicals. Toxicol In Vitro 25(7): 1425-1434.

Takahashi Y, Hayashi T, Koike M, Sakaguchi H, Kuwahara H, Nishiyama N. 2010. An interlaboratory study of the short time exposure (STE) test using SIRC cells for predicting eye irritation potential. Cutan Ocul Toxicol 29(2): 77-90.

Takahashi Y, Hayashi T, Watanabe S, Hayashi K, Koike M, Aisawa N, et al. 2009. Inter-laboratory study of short time exposure (STE) test for predicting eye irritation potential of chemicals and correspondence to globally harmonized system (GHS) classification. J Toxicol Sci 34(6): 611-626.

Takahashi Y, Koike M, Honda H, Ito Y, Sakaguchi H, Suzuki H, et al. 2008. Development of the short time exposure (STE) test: an in vitro eye irritation test using SIRC cells. Toxicol In Vitro 22(3): 760-770.

UN. 2011. Globally Harmonized System for Classification and Labelling of Chemicals (GHS). 4th rev. ed. New York:United Nations.

Documents

NICEATM Summary Review Document: Short Time Exposure Test … · 2020. 10. 19. · Short Time Exposure (STE) Test Method Summary Review Document Interagency Coordinating Committee